Sub-diffraction limit image resolution and other imaging techniques

ABSTRACT

The present invention generally relates to sub-diffraction limit image resolution and other imaging techniques. In one aspect, the invention is directed to determining and/or imaging light from two or more entities separated by a distance less than the diffraction limit of the incident light. For example, the entities may be separated by a distance of less than about 1000 nm, or less than about 300 nm for visible light. In one set of embodiments, the entities may be selectively activatable, i.e., one entity can be activated to produce light, without activating other entities. A first entity may be activated and determined (e.g., by determining light emitted by the entity), then a second entity may be activated and determined. The entities may be immobilized relative to each other and/or to a common entity. The emitted light may be used to determine the positions of the first and second entities, for example, using Gaussian fitting or other mathematical techniques, and in some cases, with sub-diffraction limit resolution. The methods may thus be used, for example, to determine the locations of two or more entities immobilized relative to a common entity, for example, a surface, or a biological entity such as DNA, a protein, a cell, a tissue, etc. The entities may also be determined with respect to time, for example, to determine a time-varying reaction. Other aspects of the invention relate to systems for sub-diffraction limit image resolution, computer programs and techniques for sub-diffraction limit image resolution, methods for promoting sub-diffraction limit image resolution, methods for producing photoswitchable entities, and the like.

RELATED APPLICATIONS

This application claims priority to all of the following according tothe following recitation of priority relationships. This application isa continuation of U.S. patent application Ser. No. 14/101,071, filedDec. 9, 2013, entitled “Sub-Diffraction Limit Image Resolution and OtherImaging Techniques,” which is a continuation of U.S. patent applicationSer. No. 12/795,423, filed Jun. 7, 2010, entitled “Sub-Diffraction LimitImage Resolution and Other Imaging Techniques,” which is a continuationof U.S. patent application Ser. No. 12/012,524, filed Feb. 1, 2008,entitled “Sub-Diffraction Limit Image Resolution and Other ImagingTechniques,” which is a continuation-in-part of International PatentApplication No. PCT/US2007/017618, filed Aug. 7, 2007, entitled“Sub-Diffraction Limit Image Resolution and Other Imaging Techniques,”which is a continuation-in-part of U.S. patent application Ser. No.11/605,842, filed Nov. 29, 2006, entitled “Sub-Diffraction ImageResolution and Other Imaging Techniques,” which claims the benefit ofU.S. Provisional Patent Application Ser. No. 60/836,167, filed Aug. 7,2006, entitled “Sub-Diffraction Image Resolution,” and the benefit ofU.S. Provisional Patent Application Ser. No. 60/836,170, filed Aug. 8,2006, entitled “Sub-Diffraction Image Resolution.” Said Ser. No.12/012,524 is also a continuation-in-part of said Ser. No. 11/605,842,which claims the benefit of said Ser. Nos. 60/836,167 and 60/836,170.Each of the above is incorporated herein by reference.

GOVERNMENT FUNDING

This invention was made with U.S. Government support under GM068518awarded by the National Institutes of Health and under N66001-04-1-8903awarded by U.S. Navy SPAWAR/SD. The U.S. Government has certain rightsin the invention.

FIELD OF INVENTION

The present invention generally relates to sub-diffraction limit imageresolution and other imaging techniques.

BACKGROUND

Fluorescence microscopy is widely used in molecular and cell biology andother applications for non-invasive, time-resolved imaging. Despitethese advantages, standard fluorescence microscopy is not useful forultra-structural imaging, due to a resolution limit set by thediffraction of light. Several approaches have been employed to try topass this diffraction limit, including near-field scanning opticalmicroscopy (NSOM), multi-photon fluorescence, stimulated emissiondepletion (STED), reversible saturable optical linear fluorescencetransition (RESOLFT), and saturated structured-illumination microscopy(SSIM), but each has certain unsatisfactory limitations. Electronmicroscopy is often used for high resolution imaging of biologicalsamples, but microscopy uses electrons, rather than light, and isdifficult to use with biological samples due to its preparationrequirements. Accordingly, new techniques are needed to harness thebenefits of fluorescence microscopy for ultra-resolution imaging ofbiological and other samples.

SUMMARY OF THE INVENTION

The present invention generally relates to sub-diffraction limit imageresolution and other imaging techniques. The subject matter of thepresent invention involves, in some cases, interrelated products,alternative solutions to a particular problem, and/or a plurality ofdifferent uses of one or more systems and/or articles.

The invention is a method, in one aspect. In one set of embodiments, themethod includes acts of providing a first entity and a second entityseparated by a distance of less than about 1000 nm, determining lightemitted by the first entity, determining light emitted by the secondentity, and determining the positions of the first entity and the secondentity by using the light emitted by the first entity and the lightemitted by the second entity. In another set of embodiments, the methodincludes acts of providing a first entity and a second entity separatedby a distance of less than about 1000 nm, activating the first entitybut not the second entity, determining light emitted by the firstentity, activating the second entity, determining light emitted by thesecond entity, and determining the positions of the first entity and thesecond entity by using the light emitted by the first entity and thelight emitted by the second entity.

The method, according to yet another set of embodiments, includes actsof providing a plurality of entities able to emit light, at least someof which are separated by a distance of less than about 1000 nm,activating a fraction of the plurality of entities to emit light,determining the emitted light, deactivating the activated fraction ofthe plurality of entities, and repeating the acts of activating anddeactivating the plurality of entities to determine the positions of theplurality of entities.

In another set of embodiments, the method includes acts of providing afirst entity and a second entity separated by a distance of separation,determining light emitted by the first entity, the light emitted by thefirst entity having a wavelength greater than the distance ofseparation, determining light emitted by the second entity, anddetermining the positions of the first entity and the second entity byusing the light emitted by the first entity and the light emitted by thesecond entity. The method, in yet another set of embodiments, includesacts of providing a first entity and a second entity separated by adistance of separation, activating the first entity but not the secondentity, determining light emitted by the first entity, the light emittedby the first entity having a wavelength greater than the distance ofseparation, activating the second entity, determining light emitted bythe second entity, and determining the positions of the first entity andthe second entity by using the light emitted by the first entity and thelight emitted by the second entity.

In one set of embodiments, the method includes acts of providing aplurality of entities able to emit light, at least some of which areseparated by a distance of separation less than the wavelength of theemitted light, activating a fraction of the plurality of entities toemit light, determining the emitted light, deactivating the activatedfraction of the plurality of entities, and repeating the acts ofactivating and deactivating the plurality of entities to determine thepositions of the plurality of entities.

The method, in another set of embodiments, includes acts of providing afirst entity and a second entity separated by a distance of less thanabout 1000 nm where the first entity and the second entity each areimmobilized relative to a common entity, determining the positions ofthe first entity and the second entity at a first point of time,determining the positions of the first entity and the second entity at asecond point of time, and determining movement and/or structural changesof the common entity using the positions of the first and secondentities at the first and second points of time. In some cases, theentities may be resolved or imaged in time. One or both of theseentities may be photoactivatable or photoswitchable in some cases. Thetwo entities may be chemically identical or distinct, for example, toallow multi-color imaging. In yet another set of embodiments, the methodincludes acts of providing a first entity and a second entity separatedby a distance of separation where the first entity and the second entityare each immobilized relative to a common entity, determining thepositions of the first entity and the second entity at a first point oftime using light emitted by the first entity and light emitted by thesecond entity where the light emitted by the first entity has awavelength greater than the distance of separation, determining thepositions of the first entity and the second entity at a second point oftime, and determining movement and/or structural changes of the commonentity using the positions of the first and second entities at the firstand second points of time. In some cases, the entities may be resolvedor imaged in time. One or both of these entities may be photoactivatableor photoswitchable in some cases. The two entities may be chemicallyidentical or distinct, for example, to allow multi-color imaging.

In still another set of embodiments, the method includes acts ofidentifying, within a series of images in time, one or morelight-emission regions, each generated by a single entity; for eachlight-emission region, identifying the center of the light-emissionregion; and for each light-emission region, reconstructing the positionof the single entity generating the light-emission region at aresolution greater than the wavelength of the light emitted by thesingle entity. Some or all of these entities may be photoactivatable orphotoswitchable. The entities may be chemically identical or distinctfor example, to allow multi-color imaging.

In another aspect, the invention is directed to an article including atranslation stage for a microscope having a drift of less than about 100nm/min, and/or an article including time-modulated light sources thatcan be switched on and off periodically and/or in a programmed fashion,and/or an article including detectors for detecting fluorescenceemission.

Still another aspect of the invention is directed to an imagingcomposition. The composition, according to one set of embodiments,includes a light-emitting entity, capable of being reversibly orirreversibly switched between a first state able to emit light at afirst, emission wavelength and a second state that does notsubstantially emit light at the first wavelength. In one embodiment, thelight-emitting entity comprises a first portion that is capable ofemitting light at the first wavelength, and a second portion thatactivates the first portion upon exposure to an external stimulus,thereby causing the first portion to emit light at the first wavelength.

In another aspect, the present invention is directed to a system forperforming one or more of the embodiments described herein. In anotheraspect, the present invention is directed to computer programs andtechniques for performing one or more of the embodiments describedherein. For example, one embodiment of the invention is directed to amachine-readable medium comprising a program, embodied in the medium,for causing a machine to perform a method comprising acts ofidentifying, within a series of images in time, one or morelight-emission regions, each generated by a single entity; for eachlight-emission region, identifying the center of the light-emissionregion; and for each light-emission region, reconstructing the positionof the single entity generating the light-emission region at aresolution greater than the wavelength of the light emitted by thesingle entity.

Other advantages and novel features of the present invention will becomeapparent from the following detailed description of various non-limitingembodiments of the invention when considered in conjunction with theaccompanying figures. In cases where the present specification and adocument incorporated by reference include conflicting and/orinconsistent disclosure, the present specification shall control. If twoor more documents incorporated by reference include conflicting and/orinconsistent disclosure with respect to each other, then the documenthaving the later effective date shall control.

BRIEF DESCRIPTION OF THE DRAWINGS

Non-limiting embodiments of the present invention will be described byway of example with reference to the accompanying figures, which areschematic and are not intended to be drawn to scale. In the figures,each identical or nearly identical component illustrated is typicallyrepresented by a single numeral. For purposes of clarity, not everycomponent is labeled in every figure, nor is every component of eachembodiment of the invention shown where illustration is not necessary toallow those of ordinary skill in the art to understand the invention. Inthe figures:

FIGS. 1A-1B illustrate the principle of sub-diffraction limit resolutionimaging, according to one embodiment of the invention, and aphotoswitchable Cy3-Cy5 moiety;

FIGS. 2A-2D illustrate the localization of molecular entities accordingto another embodiment of the invention;

FIGS. 3A-3D illustrate the sub-diffraction limit localization ofphotoswitchable molecular entities according to yet another embodimentof the invention;

FIG. 4 illustrates the repeated switching of Cy3-Cy5-labeled antibody,in accordance with one embodiment of the invention;

FIGS. 5A-5B illustrate drift correction in another embodiment of theinvention;

FIG. 6 illustrates the repeated switching of two molecular switches, inyet another embodiment of the invention;

FIGS. 7A-7E respectively illustrate the structures of Cy3, Cy5, Cy5.5,Cy7, and an example of a linked Cy3-Cy5 moiety;

FIGS. 8A-8B illustrate various photoswitchable entities, according tocertain embodiments of the invention;

FIGS. 9A-9C illustrate absorption and emission spectra of variousmoieties, according to certain embodiments of the invention;

FIGS. 10A-10F illustrate sub-diffraction limit imaging of cells withphotoswitchable entities, according to certain embodiments of theinvention;

FIGS. 11A-11C illustrate multi-color, sub-diffraction limit imaging ofcells with photoswitchable entities, according to certain embodiments ofthe invention;

FIGS. 12A-12F illustrate multi-color, sub-diffraction limit localizationof multiple types of photoswitchable molecular, entities according toyet another embodiment of the invention;

FIGS. 13A-13H illustrate various chemical structures and properties ofcertain entities of the invention;

FIG. 14 illustrates photoswitching behavior of Alexa 405-Cy7, in oneembodiment of the invention;

FIG. 15 is a conventional fluorescence image of certain DNA constructslabeled with Cy3-Cy5, Cy2-Cy5, or Alexa 405-Cy5, in an embodiment of theinvention;

FIG. 16 shows crosstalk analysis for a three-color image of a DNAsample, in another embodiment of the invention;

FIG. 17 shows localization accuracy for a single-color image of a cell,in one embodiment of the invention;

FIG. 18 shows localization accuracy for a two-color image of a cell, inanother embodiment of the invention;

FIGS. 19A-B shows images of various clathrin-coated pits, in yet anotherembodiment of the invention; and

FIGS. 20A-20F show various emissive entities containing succinimidemoieties.

DETAILED DESCRIPTION

The present invention generally relates to sub-diffraction limit imageresolution and other imaging techniques. In one aspect, the invention isdirected to determining and/or imaging light from two or more entitiesseparated by a distance less than the diffraction limit of the incidentlight. For example, the entities may be separated by a distance of lessthan about 1000 nm, or less than about 300 nm for visible light. In oneset of embodiments, the entities may be selectively activatable, i.e.,one entity can be activated to produce light, without activating otherentities. A first entity may be activated and determined (e.g., bydetermining light emitted by the entity), then a second entity may beactivated and determined. The entities may be immobilized relative toeach other and/or to a common entity. The emitted light may be used todetermine the positions of the first and second entities, for example,using Gaussian fitting or other mathematical techniques, and in somecases, with sub-diffraction limit resolution. The methods may thus beused, for example, to determine the locations of two or more entitiesimmobilized relative to (directly or indirectly, e.g., via a linker) acommon entity, for example, a surface, or a biological entity such asDNA, a protein, a cell, a tissue, a biomolecular complex, etc. Theentities may also be determined with respect to time, for example, todetermine a time-varying reaction. Other aspects of the invention relateto systems for sub-diffraction limit image resolution, computer programsand techniques for sub-diffraction limit image resolution, methods forpromoting sub-diffraction limit image resolution, methods for producingphotoswitchable entities, and the like.

In various aspects of the invention, any entity able to emit light maybe used. The entity may be a single molecule in some cases. Non-limitingexamples of emissive entities include fluorescent entities(fluorophores) or phosphorescent entities, for example, cyanine dyes(e.g., Cy2, Cy3, Cy5, Cy5.5, Cy7, etc.) metal nanoparticles,semiconductor nanoparticles or “quantum dots,” or fluorescent proteinssuch as GFP (Green Fluorescent Protein). Other light-emissive entitiesare readily known to those of ordinary skill in the art. As used herein,the term “light” generally refers to electromagnetic radiation, havingany suitable wavelength (or equivalently, frequency). For instance, insome embodiments, the light may include wavelengths in the optical orvisual range (for example, having a wavelength of between about 400 nmand about 700 nm, i.e., “visible light”), infrared wavelengths (forexample, having a wavelength of between about 300 micrometers and 700nm), ultraviolet wavelengths (for example, having a wavelength ofbetween about 400 nm and about 10 nm), or the like. In certain cases, asdiscussed in detail below, more than one entity may be used, i.e.,entities that are chemically different or distinct, for example,structurally. However, in other cases, the entities may be chemicallyidentical or at least substantially chemically identical.

In one set of embodiments, the entity is “switchable,” i.e., the entitycan be switched between two or more states, at least one of which emitslight having a desired wavelength. In the other state(s), the entity mayemit no light, or emit light at a different wavelength. For instance, anentity may be “activated” to a first state able to produce light havinga desired wavelength, and “deactivated” to a second state. An entity is“photoactivatable” if it can be activated by incident light of asuitable wavelength. As a non-limiting example, Cy5, can be switchedbetween a fluorescent and a dark state in a controlled and reversiblemanner by light of different wavelengths, i.e., 633 nm red light canswitch or deactivate Cy5 to a stable dark state, while 532 nm greenlight can switch or activate the Cy5 back to the fluorescent state. Insome cases, the entity can be reversibly switched between the two ormore states, e.g., upon exposure to the proper stimuli. For example, afirst stimuli (e.g., a first wavelength of light) may be used toactivate the switchable entity, while a second stimuli (e.g., a secondwavelength of light) may be used to deactivate the switchable entity,for instance, to a non-emitting state. Any suitable method may be usedto activate the entity. For example, in one embodiment, incident lightof a suitable wavelength may be used to activate the entity to emitlight, i.e., the entity is “photoswitchable.” Thus, the photoswitchableentity can be switched between different light-emitting or non-emittingstates by incident light, e.g., of different wavelengths. The light maybe monochromatic (e.g., produced using a laser) or polychromatic. Inanother embodiment, the entity may be activated upon stimulation byelectric field and/or magnetic field. In other embodiments, the entitymay be activated upon exposure to a suitable chemical environment, e.g.,by adjusting the pH, or inducing a reversible chemical reactioninvolving the entity, etc. Similarly, any suitable method may be used todeactivate the entity, and the methods of activating and deactivatingthe entity need not be the same. For instance, the entity may bedeactivated upon exposure to incident light of a suitable wavelength, orthe entity may be deactivated by waiting a sufficient time.

Typically, a “switchable” entity can be identified by one of ordinaryskill in the art by determining conditions under which an entity in afirst state can emit light when exposed to an excitation wavelength,switching the entity from the first state to the second state, e.g.,upon exposure to light of a switching wavelength, then showing that theentity, while in the second state can no longer emit light (or emitslight at a much reduced intensity) when exposed to the excitationwavelength.

In one set of embodiments, as discussed, a switchable entity may beswitched upon exposure to light. In some cases, the light used toactivate the switchable entity may come from an external source (e.g., alight source such as a fluorescent light source, another light-emittingentity proximate the switchable entity, etc.). The second, lightemitting entity, in some cases, may be a fluorescent entity, and incertain embodiments, the second, light-emitting entity may itself alsobe a switchable entity.

In some embodiments, the switchable entity includes a first,light-emitting portion (e.g., a fluorophore), and a second portion thatactivates or “switches” the first portion. For example, upon exposure tolight, the second portion of the switchable entity may activate thefirst portion, causing the first portion to emit light. Examples ofactivator portions include, but are not limited to, Alexa Fluor 405(Invitrogen), Alexa Fluor 488 (Invitrogen), Cy2 (GE Healthcare), Cy3 (GEHealthcare), Cy3B (GE Healthcare), Cy3.5 (GE Healthcare), or othersuitable dyes. Examples of light-emitting portions include, but are notlimited to, Cy5, Cy5.5 (GE Healthcare), Cy7 (GE Healthcare), Alexa Fluor647 (Invitrogen), Alexa Fluor 680 (Invitrogen), Alexa Fluor 700(Invitrogen), Alexa Fluor 750 (Invitrogen), Alexa Fluor 790(Invitrogen), DiD, DiR, YOYO-3 (Invitrogen), YO-PRO-3 (Invitrogen),TOT-3 (Invitrogen), TO-PRO-3 (Invitrogen) or other suitable dyes. Thesemay linked together, e.g., covalently, for example, directly, or througha linker, e.g., forming compounds such as, but not limited to, Cy5-AlexaFluor 405, Cy5-Alexa Fluor 488, Cy5-Cy2, Cy5-Cy3, Cy5-Cy3.5, Cy5.5-AlexaFluor 405, Cy5.5-Alexa Fluor 488, Cy5.5-Cy2, Cy5.5-Cy3, Cy5.5-Cy3.5,Cy7-Alexa Fluor 405, Cy7-Alexa Fluor 488, Cy7-Cy2, Cy7-Cy3, Cy7-Cy3.5,Alexa Fluor 647-Alexa Fluor 405, Alexa Fluor 647-Alexa Fluor 488, AlexaFluor 647-Cy2, Alexa Fluor 647-Cy3, or Alexa Fluor 647-Cy3.5. Thestructures of Cy3, Cy5, Cy5.5, and Cy7 are shown in FIG. 7, with anon-limiting example of a linked version of Cy3-Cy5 shown in FIG. 7E;those of ordinary skill in the art will be aware of the structures ofthese and other compounds, many of which are available commercially. Theportions may be linked via a covalent bond, or by a linker, such asthose described in detail below. Other light-emitting or activatorportions may include portions having two quaternized nitrogen atomsjoined by a polymethine chain, where each nitrogen is independently partof a heteroaromatic moiety, such as pyrrole, imidazole, thiazole,pyridine, quinoine, indole, benzothiazole, etc., or part of anonaromatic amine. In some cases, there may be 5, 6, 7, 8, 9, or morecarbon atoms between the two nitrogen atoms.

In certain cases, the light-emitting portion and the activator portions,when isolated from each other, may each be fluorophores, i.e., entitiesthat can emit light of a certain, emission wavelength when exposed to astimulus, for example, an excitation wavelength. However, when aswitchable entity is formed that comprises the first fluorophore and thesecond fluorophore, the first fluorophore forms a first, light-emittingportion and the second fluorophore forms an activator portion thatswitches that activates or “switches” the first portion in response to astimulus. For example, the switchable entity may comprise a firstfluorophore directly bonded to the second fluorophore, or the first andsecond entity may be connected via a linker or a common entity. Whethera pair of light-emitting portion and activator portion produces asuitable switchable entity can be tested by methods known to those ofordinary skills in the art. For example, light of various wavelength canbe used to stimulate the pair and emission light from the light-emittingportion can be measured to determined wither the pair makes a suitableswitch.

As a non-limiting example, Cy3 and Cy5 may be linked together to formsuch an entity. Such a procedure is described in more detail in theExamples, below. In this example, Cy3 is an activator portion that isable to activate Cy5, the light-emission portion. Thus, light at or nearthe absorption maximum (e.g., near 532 nm light for Cy3) of theactivation or second portion of the entity may cause that portion toactivate the first, light-emitting portion, thereby causing the firstportion to emit light (e.g., near 633 nm for Cy5). As previouslydescribed, the first, light-emitting portion can subsequently bedeactivated by any suitable technique (e.g., by directing 633 nm redlight to the Cy5 portion of the molecule).

Other non-limiting examples of potentially suitable activator portionsinclude 1,5 IAEDANS, 1,8-ANS, 4-Methylumbelliferone,5-carboxy-2,7-dichlorofluorescein, 5-Carboxyfluorescein (5-FAM),5-Carboxynapthofluorescein, 5-Carboxytetramethylrhodamine (5-TAMRA),5-FAM (5-Carboxyfluorescein), 5-HAT (Hydroxy Tryptamine), 5-HydroxyTryptamine (HAT), 5-ROX (carboxy-X-rhodamine), 5-TAMRA(5-Carboxytetramethylrhodamine), 6-Carboxyrhodamine 6G, 6-CR 6G, 6-JOE,7-Amino-4-methylcoumarin, 7-Aminoactinomycin D (7-AAD),7-Hydroxy-4-methylcoumarin, 9-Amino-6-chloro-2-methoxyacridine, ABQ,Acid Fuchsin, ACMA (9-Amino-6-chloro-2-methoxyacridine), AcridineOrange, Acridine Red, Acridine Yellow, Acriflavin, Acriflavin FeulgenSITSA, Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 430, Alexa Fluor488, Alexa Fluor 500, Alexa Fluor 514, Alexa Fluor 532, Alexa Fluor 546,Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 610,Alexa Fluor 633, Alexa Fluor 635, Alizarin Complexon, Alizarin Red, AMC,AMCA-S, AMCA (Aminomethylcoumarin), AMCA-X, Aminoactinomycin D,Aminocoumarin, Aminomethylcoumarin (AMCA), Anilin Blue, Anthrocylstearate, APTRA-BTC, APTS, Astrazon Brilliant Red 4G, Astrazon Orange R,Astrazon Red 6B, Astrazon Yellow 7 GLL, Atabrine, ATTO 390, ATTO 425,ATTO 465, ATTO 488, ATTO 495, ATTO 520, ATTO 532, ATTO 550, ATTO 565,ATTO 590, ATTO 594, ATTO 610, ATTO 611X, ATTO 620, ATTO 633, ATTO 635,ATTO 647, ATTO 647N, ATTO 655, ATTO 680, ATTO 700, ATTO 725, ATTO 740,ATTO-TAG CBQCA, ATTO-TAG FQ, Auramine, Aurophosphine G, Aurophosphine,BAO 9 (Bisaminophenyloxadiazole), BCECF (high pH), BCECF (low pH),Berberine Sulphate, Bimane, Bisbenzamide, Bisbenzimide (Hoechst),bis-BTC, Blancophor FFG, Blancophor SV, BOBO-1, BOBO-3, Bodipy 492/515,Bodipy 493/503, Bodipy 500/510, Bodipy 505/515, Bodipy 530/550, Bodipy542/563, Bodipy 558/568, Bodipy 564/570, Bodipy 576/589, Bodipy 581/591,Bodipy 630/650-X, Bodipy 650/665-X, Bodipy 665/676, Bodipy Fl, Bodipy FLATP, Bodipy Fl-Ceramide, Bodipy R6G, Bodipy TMR, Bodipy TMR-X conjugate,Bodipy TMR-X, SE, Bodipy TR, Bodipy TR ATP, Bodipy TR-X SE, BO-PRO-1,BO-PRO-3, Brilliant Sulphoflavin FF, BTC, BTC-5N, Calcein, Calcein Blue,Calcium Crimson, Calcium Green, Calcium Green-1 Ca²⁺ Dye, CalciumGreen-2 Ca²⁺, Calcium Green-5N Ca²⁺, Calcium Green-C18 Ca²⁺, CalciumOrange, Calcofluor White, Carboxy-X-rhodamine (5-ROX), Cascade Blue,Cascade Yellow, Catecholamine, CCF2 (GeneBlazer), CFDA, Chromomycin A,Chromomycin A, CL-NERF, CMFDA, Coumarin Phalloidin, CPM Methylcoumarin,CTC, CTC Formazan, Cy2, Cy3.1 8, Cy3.5, Cy3, Cy5.1 8, cyclic AMPFluorosensor (FiCRhR), Dabcyl, Dansyl, Dansyl Amine, Dansyl Cadaverine,Dansyl Chloride, Dansyl DHPE, Dansyl fluoride, DAPI, Dapoxyl, Dapoxyl 2,Dapoxyl 3′ DCFDA, DCFH (Dichlorodihydrofluorescein Diacetate), DDAO, DHR(Dihydorhodamine 123), Di-4-ANEPPS, Di-8-ANEPPS (non-ratio), DiA(4-Di-16-ASP), Dichlorodihydrofluorescein Diacetate (DCFH),DiD-Lipophilic Tracer, DiD (DiIC18(5)), DIDS, Dihydorhodamine 123 (DHR),DiI (DiIC18(3)), Dinitrophenol, DiO (DiOC18(3)), DiR, DiR (DiIC18(7)),DM-NERF (high pH), DNP, Dopamine, DTAF, DY-630-NHS, DY-635-NHS, DyLight405, DyLight 488, DyLight 549, DyLight 633, DyLight 649, DyLight 680,DyLight 800, ELF 97, Eosin, Erythrosin, Erythrosin ITC, EthidiumBromide, Ethidium homodimer-1 (EthD-1), Euchrysin, EukoLight, Europium(III) chloride, Fast Blue, FDA, Feulgen (Pararosaniline), FIF(Formaldehyd Induced Fluorescence), FITC, Flazo Orange, Fluo-3, Fluo-4,Fluorescein (FITC), Fluorescein Diacetate, Fluoro-Emerald, Fluoro-Gold(Hydroxystilbamidine), Fluor-Ruby, FluorX, FM 1-43, FM 4-46, Fura Red(high pH), Fura Red/Fluo-3, Fura-2, Fura-2/BCECF, Genacryl Brilliant RedB, Genacryl Brilliant Yellow 10GF, Genacryl Pink 3G, Genacryl YellowSGF, GeneBlazer (CCF2), Gloxalic Acid, Granular blue, Haematoporphyrin,Hoechst 33258, Hoechst 33342, Hoechst 34580, HPTS, Hydroxycoumarin,Hydroxystilbamidine (FluoroGold), Hydroxytryptamine, Indo-1, highcalcium, Indo-1, low calcium, Indodicarbocyanine (DiD),Indotricarbocyanine (DiR), Intrawhite Cf, JC-1, JO-JO-1, JO-PRO-1,LaserPro, Laurodan, LDS 751 (DNA), LDS 751 (RNA), Leucophor PAF,Leucophor SF, Leucophor WS, Lissamine Rhodamine, Lissamine Rhodamine B,Calcein/Ethidium homodimer, LOLO-1, LO-PRO-1, Lucifer Yellow, LysoTracker Blue, Lyso Tracker Blue-White, Lyso Tracker Green, Lyso TrackerRed, Lyso Tracker Yellow, LysoSensor Blue, LysoSensor Green, LysoSensorYellow/Blue, Mag Green, Magdala Red (Phloxin B), Mag-Fura Red,Mag-Fura-2, Mag-Fura-5, Mag-Indo-1, Magnesium Green, Magnesium Orange,Malachite Green, Marina Blue, Maxilon Brilliant Flavin 10 GFF, MaxilonBrilliant Flavin 8 GFF, Merocyanin, Methoxycoumarin, Mitotracker GreenFM, Mitotracker Orange, Mitotracker Red, Mitramycin, Monobromobimane,Monobromobimane (mBBr-GSH), Monochlorobimane, MPS (Methyl Green PyronineStilbene), NBD, NBD Amine, Nile Red, Nitrobenzoxadidole, Noradrenaline,Nuclear Fast Red, Nuclear Yellow, Nylosan Brilliant lavin EBG, OregonGreen, Oregon Green 488-X, Oregon Green, Oregon Green 488, Oregon Green500, Oregon Green 514, Pacific Blue, Pararosaniline (Feulgen), PBFI,Phloxin B (Magdala Red), Phorwite AR, Phorwite BKL, Phorwite Rev,Phorwite RPA, Phosphine 3R, PKH26 (Sigma), PKH67, PMIA, Pontochrome BlueBlack, POPO-1, POPO-3, PO-PRO-1, PO-PRO-3, Primuline, Procion Yellow,Propidium lodid (PI), PyMPO, Pyrene, Pyronine, Pyronine B, PyrozalBrilliant Flavin 7GF, QSY 7, Quinacrine Mustard, Resorufin, RH 414,Rhod-2, Rhodamine, Rhodamine 110, Rhodamine 123, Rhodamine 5 GLD,Rhodamine 6G, Rhodamine B, Rhodamine B 200, Rhodamine B extra, RhodamineBB, Rhodamine BG, Rhodamine Green, Rhodamine Phallicidine, RhodaminePhalloidine, Rhodamine Red, Rhodamine WT, Rose Bengal, S65A, S65C, S65L,S65T, SBFI, Serotonin, Sevron Brilliant Red 2B, Sevron Brilliant Red 4G,Sevron Brilliant Red B, Sevron Orange, Sevron Yellow L, SITS, SITS(Primuline), SITS (Stilbene Isothiosulphonic Acid), SNAFL calcein,SNAFL-1, SNAFL-2, SNARF calcein, SNARF1, Sodium Green, SpectrumAqua,SpectrumGreen, SpectrumOrange, Spectrum Red, SPQ(6-methoxy-N-(3-sulfopropyl)quinolinium), Stilbene, Sulphorhodamine Bcan C, Sulphorhodamine Extra, SYTO 11, SYTO 12, SYTO 13, SYTO 14, SYTO15, SYTO 16, SYTO 17, SYTO 18, SYTO 20, SYTO 21, SYTO 22, SYTO 23, SYTO24, SYTO 25, SYTO 40, SYTO 41, SYTO 42, SYTO 43, SYTO 44, SYTO 45, SYTO59, SYTO 60, SYTO 61, SYTO 62, SYTO 63, SYTO 64, SYTO 80, SYTO 81, SYTO82, SYTO 83, SYTO 84, SYTO 85, SYTOX Blue, SYTOX Green, SYTOX Orange,Tetracycline, Tetramethylrhodamine (TAMRA), Texas Red, Texas Red-Xconjugate, Thiadicarbocyanine (DiSC3), Thiazine Red R, Thiazole Orange,Thioflavin 5, Thioflavin S, Thioflavin TCN, Thiolyte, Thiozole Orange,Tinopol CBS (Calcofluor White), TMR, TO-PRO-1, TO-PRO-3, TO-PRO-5,TOTO-1, TOTO-3, TRITC (tetramethylrodamine isothiocyanate), True Blue,TruRed, Ultralite, Uranine B, Uvitex SFC, WW 781, X-Rhodamine, XRITC,Xylene Orange, Y66F, Y66H, Y66W, YO-PRO-1, YO-PRO-3, YOYO-1, YOYO-3,SYBR Green, Thiazole orange (interchelating dyes), or combinationsthereof.

Accordingly, in one embodiment of the invention, a light-emittingswitchable entity is provided, comprising a first, light emittingportion and a second, activation portion. The entity has a maximumemission wavelength determined by the first, light emitting portion anda maximum activation wavelength determined by the second, activationportion. Notably, the two wavelengths are not controlled by the samemolecular entity, and are effectively decoupled. In some cases, the samewavelength light can be used both for activating the emitting portion toa fluorescent state and for exciting emission from and deactivating theemitting portion. Further, multiple types of switchable entities withina sample may be independently determined. For example, two switchableentities having the same activator portions but different light-emissionportions can be activated by the same wavelength light applied to thesample, but emit at different wavelengths due to differentlight-emission portions and can be easily distinguished, even atseparation distances of less than sub-diffraction limit resolutions.This can effectively yield two colors in the image. Similarly, twoswitchable entities having the same light-emission portions butdifferent activator portions can be activated by different wavelengthlight applied to the sample, due to the different activator portions,and the light-emission portions may emit at same wavelengths and canthus be distinguished, even at separation distances of less thansub-diffraction limit resolutions. This also can effectively yield twocolors in the image. When these methods are combined, four (or more)color images can be readily produced. Using this principle, multi-colorimaging can be scaled up to 6 colors, 9 colors, etc., depending on theswitchable and/or activator entities. This multi-color imaging principlemay also be used with the imaging methods described herein to yieldsub-diffraction limit resolutions, and/or used to obtained multi-colorimages with other imaging methods not limited to sub-diffraction limitresolutions.

In contrast, fluorescent dyes commonly used by those of ordinary skillin the art (e.g., isolated Cy5) inherently have a maximum excitationwavelength and a maximum emission wavelength that are each determined bythe nature and structure of the fluorescent dye, and cannot beindependently controlled. Thus, the invention, in another set ofembodiments, provides a range of switchable entities where the first,light emitting portion and the second, activation portion are eachindependently selected. Accordingly, in some embodiments, a larger setof colors is provided, e.g., with respect to conventional imagingmethods.

Any suitable method may be used to link the first, light-emittingportion and the second, activation portion. For instance, thelight-emitting and the activation portions may be covalently bonded toeach other, for example, using the techniques described below. In somecases, a linker is used, and is chosen such that the distance betweenthe first and second portions is sufficiently close to allow theactivator portion to activate the light-emitting portion as desired,e.g., whenever the light-emitting portion has been deactivated in somefashion. Typically, the portions will be separated by distances on theorder of 500 nm or less, for example, less than about 300 nm, less thanabout 100 nm, less than about 50 nm, less than about 20 nm, less thanabout 10 nm, less than about 5 nm, less than about 2 nm, less than about1 nm, etc. Examples of linkers include, but are not limited to, carbonchains (e.g., alkanes or alkenes), optionally including one or moreheteroatoms, polymer units, a biological molecule such as a nucleic acid(DNA, RNA, PNA, LNA, or the like), a lipid molecule, a protein or apolypeptide, a carbohydrate or polysaccaride molecule, or the like.

In some cases, a linker comprising a rigid portion may be used. As usedherein, a “rigid” portion means a portion of a molecule, the ends ofwhich are separated by a distance which cannot change (outside of normalmolecule-scale changes in temperature, etc.) without breaking at leastone bond. Examples of rigid portions include aryl or alkyne groups. Forexample, the linker may include phenyl, pyridinyl, biphenyl, xylyl,acetylene, or the like. The light-emitting portion and/or the activatorportion may be attached to the linker using any suitable technique. Insome embodiments, the technique may include the use of attachmentsystems including an electrophile-nucleophile combination. For example,the light-emitting portion and/or the activator portion may comprise anelectrophilic atom, which refers to an atom which may be attacked by,and forms a new bond to, a nucleophilic atom (e.g., an atom having areactive pair of electrons). In some cases, the electrophilic atom maycomprise a suitable leaving group. The electrophilic atom can beattached to (e.g., reacted with) a linker comprising a nucleophilicatom. In some embodiments, the technique includes reacting alight-emitting portion and/or the activator portion comprising anucleophilic atom with a linker comprising an electrophilic atom.Non-limiting examples of functional groups comprising electrophilicatoms include a carbonyl group such as an aldehyde, an ester, acarboxylic acid, a ketone, an amide (e.g., iodoacetamide), an anhydride,an acid chloride, a hydrazone, a succinimide, a maleimide group, or analpha,beta-unsaturated ketone. Examples of functional groups comprisingnucleophilic atoms include, but are not limited to, a thiol, a hydroxylgroup, an amine, a hydrazide, or the like.

Non-limiting examples of potentially useful attachment systems includesuccinimide-amine (e.g., producing an amide), maleimide-thiol oriodoacetamide-thiol (e.g., producing a thiol ester or a thiol ether),amine-carboxylic acid, hydrazide-aldehyde or hydrazide-ketone (e.g.,producing an amine or an imine), disulfide bonds, or the like. As anexample, a light-emitting portion may contain a succinimide moiety(positioned anywhere in the light-emitting portion), and be reacted witha nucleic acid linker (e.g., DNA) containing one or more amine groups, aprotein linker (e.g., an antibody or an enzyme) containing one or moreamine groups, etc. Similarly, an activator portion may be attached tothe linker using the same, or different techniques. For instance, theactivator portion may contain a maleimide moiety, which can react with athiol on the protein linker. In some cases, such moieties can becommercially obtained. As a specific, non-limiting example, certain dyessuch as Cy3 and Cy5 are commercially sold conjugated to succinimidemoieties, and certain nucleic acids are sold modified to include variousamine groups at certain locations, such that the dyes can be conjugatedto the nucleic acids via succinimide-amine reactions. As anotherexample, an amine-modified Alexa 647, which may be obtainedcommercially, can be directly bonded to a bis-functional Cy3 NHS(N-hydroxysuccinimide) ester (also obtainable commercially) via asuccidimide-amine attachment method.

Thus, in some cases, a succinimide moiety and/or a maleimide moiety maybe attached to the light-emitting portion and/or the activator portion,and the succinimide moiety and/or maleimide moiety may be covalentlybonded to amines such as primary amines, e.g., on a linker. Non-limitingexamples of light-emitting or activators containing such moieties areshown in FIGS. 20A-20F. The light-emitting portion and/or the activatorportion may be bonded to a linker, or to each other, using suchmoieties. Examples of compounds having such moieties include thosediscussed above; structural formulae of some of these compounds can beseen in the figures. As used herein, a “succinimide moiety” is a moietyhaving a general succinimide structure, e.g.:

Similarly, a “maleimide moiety” is a moiety having a general maleimidestructure, e.g.:

where each of R¹, R², and R³ in the above structures independently is ahydrogen atom (i.e., succinimide or maleimide, respectively) orrepresents other, non-hydrogen atoms or group of atoms, for example,halogens, alkyls, alkoxyls, etc. In some cases, at least one of R¹, R²,and R³ may indicate attachment of the succinimide or maleimide moiety toa linker.

Those of ordinary skill in the art would be able to select otherattachment systems suitable for use in the context of the invention. Forexample, the light-emitting portion and/or the activator portion may beattached to the linker via pericyclic reactions (e.g., Diels-Alderreactions, cycloadditions, etc.), Wittig reactions, metal-catalyzedreactions (e.g., cross-coupling reactions), and the like.

In some cases, the light-emitting portion and/or the activator portionmay contain more than one functional group, one or more of which may beused to attach the portion to the linker. For example, thelight-emitting portion and/or the activator portion may comprise twofunctional groups (e.g., a bi-functional portion), which may be the sameor different. Those of ordinary skill in the art would be able tosynthesize compositions of the invention utilizing such bi-functional,tri-functional, or other multi-functional portions. For example, thesynthesis may comprise the use of one or more protecting groups to alterthe reactivity of one functional group relative to another functionalgroup, such that the functional groups may be reacted in a particularmanner at a selected point in the synthesis. In an illustrativeembodiment, a light-emitting portion may comprise two amine groups,where one amine may be reacted with di-tert-butyl dicarbonate to form a“protected” amine (e.g., N-tert-butoxycarbonyl- or t-BOC-protected)having reduced reactivity relative to the un-protected amine, under aparticular set of conditions. The phrase “protecting group,” as usedherein, refers to temporary substituents which protect a potentiallyreactive functional group from undesired chemical transformations.Examples of such protecting groups include esters of carboxylic acids,silyl ethers of alcohols, and acetals and ketals of aldehydes andketones, respectively. Other protecting groups are described in, forexample, Greene, T. W., Wuts, P. G. M. Protective Groups in OrganicSynthesis, 2^(nd) ed., Wiley: New York, 1991.

In one set of embodiments of the invention, the light-emittingswitchable entity can also include other switchable fluorescent probesthat do not necessarily include the two portions, such as, but notlimited to, photoactivatable or photoswitchable dye molecules, naturalor engineered fluorescent proteins that are photoactivatable orphotoswitchable, photoactivatable or photoswitchable inorganicparticles, or the like. In some cases, the switchable entity may includea first, light-emitting portion and a second, activation portion, asdiscussed herein.

In one set of embodiments, the switchable entity can be immobilized,e.g., covalently, with respect to a binding partner, i.e., a moleculethat can undergo binding with a particular analyte. Binding partnersinclude specific, semi-specific, and non-specific binding partners asknown to those of ordinary skill in the art. The term “specificallybinds,” when referring to a binding partner (e.g., protein, nucleicacid, antibody, etc.), refers to a reaction that is determinative of thepresence and/or identity of one or other member of the binding pair in amixture of heterogeneous molecules (e.g., proteins and other biologics).Thus, for example, in the case of a receptor/ligand binding pair, theligand would specifically and/or preferentially select its receptor froma complex mixture of molecules, or vice versa. Other examples include,but are not limited to, an enzyme would specifically bind to itssubstrate, a nucleic acid would specifically bind to its complement, anantibody would specifically bind to its antigen. The binding may be byone or more of a variety of mechanisms including, but not limited toionic interactions, and/or covalent interactions, and/or hydrophobicinteractions, and/or van der Waals interactions, etc. By immobilizing aswitchable entity with respect to the binding partner of a targetmolecule or structure (e.g., DNA or a protein within a cell), theswitchable entity can be used for various determination or imagingpurposes. For example, a switchable entity having an amine-reactivegroup may be reacted with a binding partner comprising amines, forexample, antibodies, proteins or enzymes. A non-limiting example of theimmobilization of a switchable entity to an antibody is discussed in theExamples, below.

In some embodiments, more than one switchable entity may be used, andthe entities may be the same or different. In some cases, the lightemitted by a first entity and the light emitted by a second entity havethe same wavelength. The entities may be activated at different timesand the light from each entity may be determined separately. This allowsthe location of the two entities to be determined separately and, insome cases, the two entities may be spatially resolved, as discussed indetail below, even at distances of separation that are less than thelight emitted by the entities or below the diffraction limit of theemitted light (i.e., “sub-diffraction limit” resolutions). In certaininstances, the light emitted by a first entity and the light emitted bya second entity have different wavelengths (for example, if the firstentity and the second entity are chemically different, and/or arelocated in different environments). The entities may be spatiallyresolved even at distances of separation that are less than the lightemitted by the entities or below the diffraction limit of the emittedlight. In certain instances, the light emitted by a first entity and thelight emitted by a second entity have substantially the samewavelengths, but the two entities may be activated by light of differentwavelengths and the light from each entity may be determined separately.The entities may be spatially resolved even at distances of separationthat are less than the light emitted by the entities, or below thediffraction limit of the emitted light.

In some embodiments, the first, light-emitting portion and the second,activation portion as described above may not be directly covalentlybonded or linked via a linker, but are each immobilized relative to acommon entity. In other embodiments, two or more of the switchableentities (some of which can include, in certain cases, a first,light-emitting portion and a second, activation portion linked togetherdirectly or through a linker) may be immobilized relative to a commonentity in some aspects of the invention. The common entity in any ofthese embodiments may be any nonbiological entity or biological entity,for example, a cell, a tissue, a substrate, a surface, a polymer, abiological molecule such as a nucleic acid (DNA, RNA, PNA, LNA, or thelike), a lipid molecule, a protein or a polypeptide, or the like, abiomolecular complex, or a biological structure, for example, anorganelle, a microtubule, a clathrin-coated pit, etc. The common entitymay accordingly be determined in some fashion, e.g., imaged. As anotherexample, two or more entities may be immobilized relative to a DNAstrand or other nucleic acid strand (e.g., using antibodies,substantially complementary oligonucleotides labeled with one or moreentities, chemical reactions or other techniques known to those ofordinary skill in the art), and their locations along the stranddetected. In some cases, the number of base pairs (bp) separating theentities along the nucleic acid strand may be determined.

In some cases, the entities may be independently switchable, i.e., thefirst entity may be activated to emit light without activating a secondentity. For example, if the entities are different, the methods ofactivating each of the first and second entities may be different (e.g.,the entities may each be activated using incident light of differentwavelengths). As another non-limiting example, incident light having asufficiently weak intensity may be applied to the first and secondentities such that only a subset or fraction of the entities within theincident light are activated, i.e., on a stochastic or random basis.Specific intensities for activation can be determined by those ofordinary skill in the art using no more than routine skill. Byappropriately choosing the intensity of the incident light, the firstentity may be activated without activating the second entity.

The second entity may be activated to emit light, and optionally, thefirst entity may be deactivated prior to activating the second entity.The second entity may be activated by any suitable technique, aspreviously described, for instance, by application of suitable incidentlight.

In some cases, incident light having a sufficiently weak intensity maybe applied to a plurality of entities such that only a subset orfraction of the entities within the incident light are activated, e.g.,on a stochastic or random basis. The amount of activation may be anysuitable fraction, e.g., about 5%, about 10%, about 15%, about 20%,about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%,about 90%, or about 95% of the entities may be activated, depending onthe application. For example, by appropriately choosing the intensity ofthe incident light, a sparse subset of the entities may be activatedsuch that at least some of them are optically resolvable from each otherand their positions can be determined. Iterative activation cycles mayallow the positions of all of the entities, or a substantial fraction ofthe entities, to be determined. In some cases, an image withsub-diffraction limit resolution can be constructed using thisinformation.

The two or more entities may be resolved, even at distances ofseparation that are less than the wavelength of the light emitted by theentities or below the diffraction limit of the emitted light, accordingto another aspect of the invention. The resolution of the entities maybe, for instance, on the order of 1 micrometer (1000 nm) or less, asdescribed herein. For example, if the emitted light is visible light,the resolution may be less than about 700 nm. In some cases, two (ormore) entities may be resolved even if separated by a distance of lessthan about 500 nm, less than about 300 nm, less than about 200 nm, lessthan about 100 nm, less than about 80 nm, less than about 60 nm, lessthan about 50 nm, or less than about 40 nm. In some cases, two or moreentities separated by a distance of at least about 20 nm or less than 10nm can be resolved using embodiments of the present invention.

Light emitted by each of the switchable entities may be determined,e.g., as an image or matrix. For example, the first entity may beactivated and the light emitted by the first entity determined, and thesecond entity may be activated (with or without deactivating the firstentity) and light emitted by the second entity may be determined. Thelight emitted by each of the plurality of entities may be at the same ordifferent wavelengths. Any suitable method may be used to determine theemitted light. For instance, a camera such as a CCD camera, aphotodiode, a photomultiplier, or a spectrometer may be used; those ofordinary skill in the art will know of other suitable techniques.Additional non-limiting example of a suitable technique for determininglight produced by the entities is discussed in the Examples, below. Insome cases, multiple images (or other determinations) may be used, forexample, to improve resolution and/or to reduce noise. For example, atleast 2, at least 5, at least 10, at least 20, at least 25, at least 50,at least 75, at least 100, etc. images may be determined, depending onthe application.

In some aspects, the light may be processed to determine the spatialpositions of the two or more entities. In some cases, the positions ofone or more entities, distributed within an image, may each beindividually determined. In one set of embodiments, the emitted lightmay be processed, using Gaussian fitting or other suitable techniques,to localize the position of each of the emissive entities. Details ofone suitable Gaussian fit technique are described in the Examples,below; those of ordinary skill in the art will be able to identify othersuitable image-processing techniques with the benefit of the presentdisclosure.

Another example of an image-processing technique follows, in accordancewith another embodiment of the invention. Starting with a series ofimages of a sample (e.g., a movie), each light-emission peak (e.g.,through fluorescence, phosphorescence, etc.) is identified, and thetimes which the peak is present are determined. For example, a peak maybe present with approximately the same intensity in a series of imageswith respect to time. Peaks may be fit, in some cases, to Gaussianand/or elliptical Gaussian functions to determine their centroidpositions, intensities, widths, and/or ellipticities. Based on theseparameters, peaks which are too dim, too wide, too skewed, etc. to yieldsatisfactory localization accuracy may be rejected in certain cases fromfurther analysis. Peaks which are sporadic, moving, discontinuouslypresent, etc. may also be discarded. By determining the center positionof the peak, for example, using least-squares fitting to a 2-dimensionalGaussian function of the peak intensities, the location of the source ofthe peak (e.g., any entity or entities able to emit light, as discussedherein) can be determined. This process may be repeated as necessary forany or all of the peaks within the sample.

In another non-limiting example of a suitable imaging processingtechnique of the present invention, a series of images of a sample (e.g.a movie) may include a repetitive sequence of activation frames (e.g.,in which the activation light is on) and imaging frames (e.g., in whichthe imaging light is on). For one or more of the imaging frames,fluorescent peaks can be identified and fit to Gaussian and/orelliptical Gaussian functions to determine their centroid positions,intensities, widths, and/or ellipticities. Based on these parameters,peaks that are too dim, too wide, too skewed, etc. to yield satisfactorylocalization accuracy may be rejected from further analysis. Peaks whichare sporadic, moving, discontinuously present, etc. may also bediscarded in some cases. By determining the center position of the peak,for example, using least-squares fitting to a 2-dimensional Gaussianfunction of the peak intensities, the location of the source of the peak(e.g., any entity or entities able to emit light, as discussed herein)can be determined. Peaks appearing in an imaging frame immediately afteran activation frame can be recognized as a controlled activation eventand may be color-coded according to the activation laser color, in someembodiments. This process may also be repeated as necessary for any orall of the peaks within the sample.

Other image-processing techniques may also be used to facilitatedetermination of the entities, for example, drift correction or noisefilters may be used. Generally, in drift correction, for example, afixed point is identified (for instance, as a fiduciary marker, e.g., afluorescent particle may be immobilized to a substrate), and movementsof the fixed point (i.e., due to mechanical drift) are used to correctthe determined positions of the switchable entities. In another examplemethod for drift correction, the correlation function between imagesacquired in different imaging frames or activation frames can becalculated and used for drift correction. In some embodiments, the driftmay be less than about 1000 nm/min, less than about 500 nm/min, lessthan about 300 nm/min, less than about 100 nm/min, less than about 50nm/min, less than about 30 nm/min, less than about 20 nm/min, less thanabout 10 nm/min, or less than 5 nm/min. Such drift may be achieved, forexample, in a microscope having a translation stage mounted for x-ypositioning of the sample slide with respect to the microscopeobjective. The slide may be immobilized with respect to the translationstage using a suitable restraining mechanism, for example, spring loadedclips. In addition, a buffer layer may be mounted between the stage andthe microscope slide. The buffer layer may further restrain drift of theslide with respect to the translation stage, for example, by preventingslippage of the slide in some fashion. The buffer layer, in oneembodiment, is a rubber or polymeric film, for instance, a siliconerubber film. Accordingly, one embodiment of the invention is directed toa device, comprising a translation stage, a restraining mechanism (e.g.,a spring loaded clip) attached to the translation stage able toimmobilize a slide, and optionally, a buffer layer (e.g., a siliconerubber film) positioned such that a slide restrained by the restrainingmechanism contacts the buffer layer.

Multiple locations on a sample may each be analyzed to determine theentities within those locations. For example, a sample may contain aplurality of various entities, some of which are at distances ofseparation that are less than the wavelength of the light emitted by theentities or below the diffraction limit of the emitted light. Differentlocations within the sample may be determined (e.g., as different pixelswithin an image), and each of those locations independently analyzed todetermine the entity or entities present within those locations. In somecases, the entities within each location may be determined toresolutions that are less than the wavelength of the light emitted bythe entities or below the diffraction limit of the emitted light, aspreviously discussed.

As noted, in some embodiments, more than one type of switchable entitymay be used in a sample, and the positions of each of the entities maybe independently determined, in some cases, at sub-diffraction limitresolutions, e.g., to generate a multi-color image with sub-diffractionlimit resolution. For instance, by repeatedly activating anddeactivating particular switchable entities in a sample, the positionsof a plurality of switchable entities may be determined, in some cases,to resolutions that are less than the wavelength of the light emitted bythe entities or below the diffraction limit of the emitted light.

As a non-limiting example, a sample may contain two types of lightemitting portions (e.g., Cy5 and Cy5.5) and two types of activationportions (e.g., Cy3 and Cy2), for a total of four (2×2) types ofswitchable entities: Cy5-Cy3, Cy5-Cy2, Cy5.5-Cy3, and Cy5.5-Cy2. TheCy3-containing entities can be activated by applying light at a suitablewavelength without activating the Cy2-containing entities (sincedifferent activation wavelengths are required) while light emitted bythe Cy5-containing molecules can be distinguished from light emitted bythe Cy5.5-containing molecules (which emits light at a differentwavelength). Thus, only the Cy5-Cy3 entities will be determined, whilethe other entities are either not activated, or are activated but thelight emitted by those entities is not used. By using theabove-described techniques, the positions of the Cy5-Cy3 entities may bedetermined within a sample, even to resolutions that are less than thewavelength of the light emitted by Cy5. Additionally, by repeating thisprocedure using suitable activation and emission wavelengths, thepositions of the other entities may also be determined, e.g., tosub-diffraction limit resolutions. In another example, the sample mayinclude three (or more) types of light emitting portions (e.g., Cy5,Cy5.5 and Cy7) and/or three (or more) types of activation portions(e.g., Alexa Fluor 405, Cy2 and Cy3). Accordingly, multi-color imaging(e.g., 4 colors, 6 colors, 8 colors, 9 colors, 10 colors, 12 colors,etc.) with sub-diffraction limit resolutions may be realized.

In some embodiments of the invention, the entities may also be resolvedas a function of time. For example, two or more entities may be observedat various time points to determine a time-varying process, for example,a chemical reaction, cell behavior, binding of a protein or enzyme, etc.Thus, in one embodiment, the positions of two or more entities may bedetermined at a first point of time (e.g., as described herein), and atany number of subsequent points of time. As a specific example, if twoor more entities are immobilized relative to a common entity, the commonentity may then be determined as a function of time, for example,time-varying processes such as movement of the common entity, structuraland/or configurational changes of the common entity, reactions involvingthe common entity, or the like. The time-resolved imaging may befacilitated in some cases since a switchable entity can be switched formultiple cycles, each cycle give one data point of the position of theentity.

Another aspect of the invention is directed to a computer-implementedmethod. For instance, a computer and/or an automated system may beprovided that is able to automatically and/or repetitively perform anyof the methods described herein. As used herein, “automated” devicesrefer to devices that are able to operate without human direction, i.e.,an automated device can perform a function during a period of time afterany human has finished taking any action to promote the function, e.g.by entering instructions into a computer. Typically, automated equipmentcan perform repetitive functions after this point in time. Theprocessing steps may also be recorded onto a machine-readable medium insome cases.

Still another aspect of the invention is generally directed to a systemable to perform one or more of the embodiments described herein. Forexample, the system may include a microscope, a device for activatingand/or switching the entities to produce light having a desiredwavelength (e.g., a laser or other light source), a device fordetermining the light emitted by the entities (e.g., a camera, which mayinclude color-filtering devices, such as optical filters), and acomputer for determining the spatial positions of the two or moreentities. In some cases, mirrors (such as dichroic mirror or apolychroic mirror), prisms, lens, diffraction gratings, or the like maybe positioned to direct light from the light source. In some cases, thelight sources may be time-modulated (e.g., by shutters, acoustic opticalmodulators, or the like). Thus, the light source may be one that isactivatable and deactivatable in a programmed or a periodic fashion. Inone embodiment, more than one light source may be used, e.g., which maybe used to illuminate a sample with different wavelengths or colors. Forinstance, the light sources may emanate light at different frequencies,and/or color-filtering devices, such as optical filters or the like maybe used to modify light coming from the light sources such thatdifferent wavelengths or colors illuminate a sample.

In some embodiments, a microscope may be configured so to collect lightemitted by the switchable entities while minimizing light from othersources of fluorescence (e.g., “background noise”). In certain cases,imaging geometry such as, but not limited to, atotal-internal-reflection geometry a spinning-disc confocal geometry, ascanning confocal geometry, an epi-fluorescence geometry, etc., may beused for sample excitation. In some embodiments, a thin layer or planeof the sample is exposed to excitation light, which may reduceexcitation of fluorescence outside of the sample plane. A high numericalaperture lens may be used to gather the light emitted by the sample. Thelight may be processed, for example, using filters to remove excitationlight, resulting in the collection of emission light from the sample. Insome cases, the magnification factor at which the image is collected canbe optimized, for example, when the edge length of each pixel of theimage corresponds to the length of a standard deviation of a diffractionlimited spot in the image.

In some cases, a computer may be used to control excitation of theswitchable entities and the acquisition of images of the switchableentities. In one set of embodiments, a sample may be excited using lighthaving various wavelengths and/or intensities, and the sequence of thewavelengths of light used to excite the sample may be correlated, usinga computer, to the images acquired of the sample containing theswitchable entities. For instance, the computer may apply light havingvarious wavelengths and/or intensities to a sample to yield differentaverage numbers of activated switchable elements in each region ofinterest (e.g., one activated entity per location, two activatedentities per location, etc). In some cases, this information may be usedto construct an image of the switchable entities, in some cases atsub-diffraction limit resolutions, as noted above.

In other aspects of the invention, the systems and methods describedherein may also be combined with other imaging techniques known to thoseof ordinary skill in the art, such as high-resolution fluorescence insitu hybridization (FISH) or immunofluorescence imaging, live cellimaging, confocal imaging, epi-fluorescence imaging, total internalreflection fluorescence imaging, etc.

The following are incorporated herein by reference: U.S. ProvisionalPatent Application Ser. No. 60/836,167, filed Aug. 7, 2006, entitled“Sub-Diffraction Image Resolution”; and U.S. Provisional PatentApplication Ser. No. 60/836,170, filed Aug. 8, 2006, entitled“Sub-Diffraction Image Resolution.”

The following examples are intended to illustrate certain embodiments ofthe present invention, but do not exemplify the full scope of theinvention.

EXAMPLES

This example shows a high-resolution optical microscopy, stochasticoptical reconstruction microscopy (“STORM”), in which a fluorescenceimage is constructed from high-accuracy localization of individualfluorescent entities (“fluorophores”) that are switched on and off usinglight of different colors, in accordance with one embodiment of theinvention. The STORM imaging process in this example includes a seriesof imaging cycles (FIG. 1A). In each cycle, a fraction of thefluorophores in the field of view are switched on or activated, suchthat each of the active fluorophores is optically resolvable from therest, i.e. their images are not overlapping. This allows the position ofthese fluorophores to be determined with high accuracy. Repeating thisprocess for multiple cycles, each causing a stochastically differentsubset of fluorophores to be turned on or activated, enables thepositions of many fluorophores to be determined and thus an overallimage to be reconstructed. In these examples, an imaging resolution ofapproximately 20 nm is demonstrated, an improvement of more than 10times over the resolution of conventional fluorescence microscopy, usinga simple total-internal-reflection fluorescence microscope, low-powercontinuous-wave lasers, and a photoswitchable cyanine dye.

FIG. 1A shows that a STORM imaging sequence using a hypotheticalhexameric object labeled with fluorophores can be switched between afluorescent and a dark state by a green and a red laser, respectively.In this non-limiting illustration, all fluorophores were first switched“off” to the dark state (“deactivated”) by a strong red laser pulse. Ineach imaging cycle, a green laser pulse was used to switch on(“activate”) only a fraction of the fluorophores to give an opticallyresolvable set of active fluorophores. Next, under red illumination(“image”), these molecules emitted fluorescence until they were switchedoff, allowing their positions (indicated by white crosses) to bedetermined with relatively high accuracy. The overall image was thenreconstructed from the fluorophore positions obtained from multipleimaging cycles.

Cyanine dye, Cy5, can be switched between a fluorescent and a dark statein a controlled and reversible manner by light of different wavelengths.Red laser light that produces fluorescent emission from Cy5 can alsoswitch the dye to a stable dark state. Exposure to green laser lightconverts Cy5 back to the fluorescent state, but the recovery rate maydepend in some cases on the close proximity of a secondary dye, Cy3. TheCy3-Cy5 dye pair can also be referred to as a switch. Under illuminationconditions allowing single-molecule detection, it was found that such aswitch, when attached or immobilized to nucleic acids or proteins, canbe cycled on and off hundreds of times before permanent photobleachingoccurs (FIG. 1B and FIG. 4). In FIG. 1B, a red laser (633 nm, 30 W/cm²)was used to excite fluorescence (black line) from Cy5 and to switch Cy5to the dark state. A green laser (532 nm, 1 W/cm²) was used to returnCy5 to the fluorescent state. The alternating red and green lineindicates the laser excitation pattern. In some cases, the recovery rateof Cy5 appeared to depend on the close proximity of Cy3.

FIG. 4 shows goat anti-mouse secondary antibody labeled with the cyanineswitch exhibits photoswitching behavior similar to switch-labeled DNA.The antibody was labeled with Cy3 and Cy5 (see below) and bound to aquartz slide coated with unlabeled mouse anti-transferrin primaryantibody. The trace shows the Cy5 fluorescence intensity detected from asingle labeled antibody as it switched on and off until permanentphotobleaching occurs after about 230 seconds. The sample was excitedwith a sequence of alternating green and red laser pulses (0.5 s greenfollowed by 2 s red).

Other photoswitchable dye pairs can be constructed using similarprinciples. For instance, in addition to Cy5, Cy5.5, Cy7, and AlexaFluor 647 can also be switched between a fluorescent and a dark state ina controlled and reversible manner by light of different wavelengths.When paired with Cy3, upon illumination with a red laser (633 nm or 657nm), each of these four dyes (Cy5, Cy5.5, Cy7, and Alexa Fluor 647) wasinitially fluorescent and then quickly switched into a non-fluorescent,dark state. A brief exposure to a green laser pulse (532 nm) led toreactivation of these dyes back to the fluorescent state (see FIGS. 8Aand 9). Furthermore, different activating portion can be used toactivate the same emitting portion in some cases. For instance, usingCy5 as an example for the emitting portion, Cy3, Cy2, and Alexa Fluor405 could be paired with Cy5. Upon illumination with a red laser (633 nmor 657 nm), Cy5 was observed to be quickly switched into the dark state.In this example, the reactivation of Cy5 required different coloredlasers corresponding to the absorption wavelength of the activator(FIGS. 8B and 9). The Alexa 405-Cy5 pair was efficiently activated by aviolet laser (405 nm), but appeared to be less sensitive to blue (457nm) and green (532 nm) lasers. Similarly, the Cy2-Cy5 pair was moresensitive to the blue light than to violet or green light, whereas theCy3-Cy5 pair was appeared more sensitive to the green laser.

Examples of activator portions include, but are not limited to, AlexaFluor 405, Alexa Fluor 488, Cy2, Cy3, Cy3.5, or Cy5. Examples oflight-emitting portions include, but are not limited to, Cy5, Cy5.5,Cy7, Alexa Fluor 647, Alexa Fluor 680, or Alexa Fluor 700. These may belinked together to form photoswitchable entities such as, but notlimited to, Cy5-Alexa Fluor 405, Cy5-Alexa Fluor 488, Cy5-Cy2, Cy5-Cy3,Cy5-Cy3.5, Cy5.5-Alexa Fluor 405, Cy5.5-Alexa Fluor 488, Cy5.5-Cy2,Cy5.5-Cy3, Cy5.5-Cy3.5, Cy7-Alexa Fluor 405, Cy7-Alexa Fluor 488,Cy7-Cy2, Cy7-Cy3, Cy7-Cy3.5, or Cy7-Cy5.

In this example, the concept of STORM is shown using the Cy5-053 switch,but any suitably optically switched fluorophores could also be used. Theresolution of STORM may be limited in some cases by the accuracy withwhich individual switches can be localized during a switching cycle. Asan example of determining the localization accuracy, a switch wasattached or immobilized to a short double-stranded DNA, which wassurface-immobilized at low density so that single switches wereresolvable. These switches were periodically cycled on and off usinggreen and red laser light, and the red laser also served to excitefluorescence from Cy5. No fluorescence from Cy3 was recorded in thisprocess. The high localization accuracy of individual switches duringeach switching cycle defining the intrinsic resolution of STORM is shownin FIG. 2. The fluorescence image from a single switch gave apoint-spread function shown in FIG. 2A. The point spread function (PSF)of the emission from a single switch on DNA during a single switchingcycle. A Gaussian fit to this image (not shown) was used to localize thecentroid position of the PSF, indicating the position of the switch. Thepositions determined from multiple switching cycles showed a substantialspread (FIG. 2B), which was significantly reduced by correcting forsample drift over the course of the experiment (FIG. 2C, see below foradditional details). FIGS. 2B and 2C show the centroid positions of anindividual switch determined in 20 successive imaging cycles before(FIG. 2B) and after (FIG. 2C) correction for sample drift. The scalebars are 20 nm. The standard deviation of the drift-corrected positionsobtained from 20 imaging cycles was on average 8 nm for individualswitches (FIG. 2D). FIG. 2D shows a histogram of the standard deviationof centroid positions. The standard deviation is determined as(σ_(x)+σ_(y))/2 ((sigma-x+sigma-y)/2) for each switch using 20 imagingcycles, where σ_(x) (sigma-x) and σ_(y) (sigma-y) are the standarddeviations of the centroid positions in the x and y dimensions. Thishistogram was constructed from 29 switches. This value gave a measure ofthe uncertainty in the localization of a single switch per imagingcycle. Correspondingly, the experimentally measured spread of switchpositions follows a Gaussian distribution with a FWHM of 18 nm, asexpected from the 8 nm standard deviation (FIG. 5). Therefore, underthese imaging conditions, two switches separated by 20 nm could beresolved.

FIG. 5 shows the superimposed position distributions of 29 individualswitches before (FIG. 5A) and after (FIG. 5B) correction for stagedrift. The scale bar is 20 nm. To obtain these distributions, the imageof each switch during a given imaging cycle was fit to a two-dimensionalGaussian function, yielding the centroid position of each switch duringthat cycle. Twenty imaging cycles were carried out, giving twentymeasured positions per switch. The position data for each switch wererealigned so that the average position of each switch was at the origin,and the measurements from different switches were then superimposed togive the overall position distribution shown in the insets. The mainplots are histograms of these centroid positions. The uncorrecteddistribution is fairly broad and skewed asymmetrically. After correctionfor stage drift using fiducial markers (see below) the distributionbecome significantly narrower. The fit of the drift-corrected histogramto a Gaussian gave a full width at half-maximum of 18±2 nm.

To demonstrate the capability of STORM to resolve fluorescent moleculesin close proximity, standard samples of linear, double-stranded DNAlabeled were constructed with multiple switches separated by awell-defined number of base pairs (see methods, below, for details). TheDNA strands were also labeled with multiple biotins and attached to ahigh-density streptavidin layer (see below), increasing the likelihoodof multiple attachments between the DNA and the surface so that the DNAwas immobilized in the plane. DNA strands were first examined containingtwo switches separated by 135 base-pairs (FIG. 3A and FIG. 6),corresponding to a length of 46 nm along the DNA contour. A STORM imageobtained using the imaging procedure described above (see below fordetails) showed two clusters of measured switch positions, indicatingthat the two switches were well-resolved (FIG. 3A). This image shows twoseparated clusters of measured switch positions (crosses), eachcorresponding to a single switch. The center-of-mass position of eachcluster is marked by a dot. The inter-switch distances are 46 nm, 44 nmand 34 nm for these three examples. The scale bars are 20 nm. Thedistance between the centers of the two clouds had a mean value of 41 nm(FIG. 3B), in quantitative agreement with the theoretical mean (40 nm)determined using the known contour and persistence lengths of the DNAsample (see below). FIG. 3B shows a comparison between the inter-switchdistances measured using STORM (grey column) and the predicted distancedistribution considering the flexibility of DNA (dashed line). FIG. 3Cshows STORM images of four switches attached to a double-stranded DNA,pair-wise separated by a contour length of 46 nm. The measured switchpositions were clustered by an automated algorithm (see below fordetails) and different clusters are indicated by different symbols. Thescale bars are 20 nm.

FIG. 6 is an excerpt of a fluorescence trace (black line) showing thecycling of Cy3-Cy5 switches separated by a contour length of 46 nm ondsDNA (see also FIG. 3A). Each switching cycle lasted for 5 seconds andincluded a brief green pulse (underlying tick marks) to activate one ortwo switches followed by a long red exposure (underlying bar) to exciteCy5 fluorescence and return the switches to the off state. In cycleswhen one switch is activated (e.g. 225-230 seconds), a single intensitylevel was apparent before the return to the dark state. In cycles wheretwo switches are activated (e.g. 245-250 seconds), a “staircase” withtwo decreasing levels corresponding to the sequential switching off ofeach dye is observed. Regions where only a single switch is on and allof the peak quality criteria are satisfied (see below) were used forsingle-switch localization.

Longer DNA samples labeled with four switches evenly separated by 46 nmalong the contour were also imaged. The STORM images revealed fourclusters of switch positions following a bent contour consistent withthe persistence length of DNA and the engineered separation between theswitches. These results indicated that STORM can image biologicalsamples with sub-diffraction limit resolution and that featuresseparated by 40 nm are well within the resolving power.

One advantage of STORM is its ability to localize a large number ofswitches within a diffraction-limited spot by cycling the switches onand off in a controlled manner, allowing this to be used as a generalbiological imaging technique. To demonstrate this capability, circularDNA plasmids were prepared and coated with RecA protein and imaged usingindirect immunofluorescence with switch-labeled secondary antibody (FIG.3D, see methods, below, for details). Here the photoswitch comprise ofCy3 and Cy5. Cy3 serve as the activating portion and Cy5 serve as thelight-emitting portion. STORM images of the RecA filaments revealedtheir circular structure with greatly increased resolution when comparedwith conventional wide-field images. Parts of the filament appear to beunlabeled or kinked, which may correspond to regions of the plasmid thatremain uncoated by RecA. In FIG. 3D, the top panels show indirectimmunofluorescence images with switch-labeled secondary antibody takenby a total internal reflection microscope. The bottom panels are thereconstructed STORM images of the same filaments. The scale bars are 300nm.

The presence of multiple types of photoswitchable pairs of lightemitting portion and activating portion may also allow multi-color STORMimaging, e.g., as described herein. This example uses a selectiveactivation scheme as an initial demonstration of multicolor STORMimaging. Three different DNA constructs labeled with Alexa 405-Cy5,Cy2-Cy5, or Cy3-Cy5 were mixed and immobilized on a microscope slide ata high surface density such that individual DNA molecules could not beresolved in a conventional fluorescence image. To generate a STORMimage, the sample was first exposed to a red laser (633 nm) to switchoff nearly all Cy5 dyes in the field of view. The sample was thenperiodically excited with a sequence of violet (405 nm), blue (457 nm),and green (532 nm) laser pulses, each of which activated a sparse,optically resolvable subset of fluorophores. In between activationpulses, the sample was imaged with the red laser. The image of eachactivated fluorescent spot was analyzed to determine its centroidposition (referred to as a localization), and a color was assignedaccording to the preceding activation pulse. As the same imaging laserand detection channel were used for all three dye pairs, there was noneed for correction of chromatic aberration. After thousands ofactivation cycles, a STORM image was constructed by plotting all of thecolored localizations (FIGS. 12A-12C). The STORM image showed separatedclusters of localizations. Each cluster corresponded to an individualDNA molecule and resulted from the repetitive localization of a singleCy5 molecule over multiple switching cycles. The majority of thelocalizations within each cluster displayed the same color, identifyingthe type of activator dye present on the DNA. There were two origins ofcrosstalk between colors that were identified: false activation by laserpulses of incorrect colors and non-specific activation by the redimaging laser, but both effects were quantitatively small. Thelocalizations within each cluster approximately follow a Gaussiandistribution with a full-width-half-maximum (FWHM) of 26±1 nm, 25±1 nm,and 24±1 nm for the three color channels (FIGS. 12D-12F), suggesting animaging resolution of ˜25 nm. This resolution is lower than thetheoretical limit predicted from the number of photons detected.

STORM imaging can also be performed on cell samples. To demonstrate thiscapability, in this example, single-color immunofluorescence imaging ofmicrotubules, which are filamentous cytoskeleton structures importantfor many cellular functions, were performed. BS-C-1 cells were fixed andimmunostained with primary antibodies against microtubules and then witha switch-labeled secondary antibody. Here, the photoswitch usedcomprised Cy3 and Alexa Fluor 647, with Cy3 serving as the activatingportion and Alexa Fluor 647 serving as the light-emitting portion. TheSTORM image showed a drastic improvement in the resolution of themicrotubule network as compared to the conventional fluorescence image(FIG. 10). FIGS. 10A, 10C, and 10E are conventional images ofmicrotubules in regions of the cell, shown at different scales, andFIGS. 10B, 10D, and 10F are STORM images of the same regions. In theregions where microtubules were densely packed and undefined in theconventional image, individual microtubule filaments were resolved bySTORM (FIGS. 10C-F). Whole-cell STORM images, including ˜10⁶single-molecule localizations, were acquired in 2 to 30 minutes.Super-resolution microtubule structures began to emerge after only about10 to 20 seconds of STORM imaging, although this was not optimized. Theeffective imaging resolution was affected both by the intrinsic dyelocalization accuracy and the size of the antibody labels. Improvementin the effective resolution may be achieved by using directimmunofluorescence staining with dye-labeled primary antibodies or Fabfragments.

Using different photoswitchable pairs, multi-color STORM imaging canalso be performed on cell samples. To demonstrate this capability,microtubules and clathrin-coated pits (CCPs), cellular structures usedfor receptor-mediated endocytosis, were simultaneously imaged. Themicrotubules and clathrin were immunostained with primary antibodies andthen with switch-labeled secondary antibodies. Here, the photoswitchescomprised Cy2 and Alexa Fluor 647 for microtubule imaging, and Cy3 andAlexa Fluor 647 for clathrin imaging, with Cy2 or Cy3 serving as theactivating portion and Alexa Fluor 647 serving as the light-emittingportion. The 457 nm and 532 nm lasers were used to selectively activatethe two pairs. Crosstalk between the two color channels due to false andnon-specific activations were subtracted from the image afterstatistical analysis. FIG. 11 shows two-color super-resolution STORMimages of microtubules and clathrin-coated pits. FIG. 11 shows thetwo-color STORM image presented at different scales. The green channel(457 nm activation) revealed filamentous structures for microtubules(long thin structures in these images). The red channel revealspredominantly spherical structures in these images, which were theclathrin-coated pits and vesicles.

In summary, this example demonstrates that STORM is capable of imagingbiological structures with sub-diffraction limit resolution. Theresolution of the technique is not limited to the wavelength of light.For the Cy3-Cy5 switch, approximately 3,000 photons were detected perswitching cycle, independent of red laser intensity (data not shown),predicting a theoretical localization accuracy of 4 nm. The differencebetween this theoretical prediction and the measured accuracy of 8 nm isrelatively small, and the discrepancy may be due to imperfect correctionof stage drift and aberration due to focus drift in the measurements.This measured localization accuracy corresponds to an imaging resolutionof approximately 20 nm (full width half maximum, FWHM). Indeed,fluorescent switches separated by ˜40 nm were readily resolved (FIG. 3).

The cyanine switches could be turned on and off reliably for hundreds ofcycles before photobleaching, allowing STORM to be used for resolvingstructures with many fluorophores in a potentially time-resolved manner.The circular structure of RecA filaments containing 10-20 switches andmicrotubule and clathrin-coated pits structures in cells could beresolved in a few minutes or less. The imaging speed may be improved,for instance, by increasing the switching rate through strongerexcitation or fluorophores with faster switching kinetics, by using acamera with a faster frame rate, by using a fast camera reading scheme,such as binning pixels or only reading out a fraction of the pixels, orby other technique. STORM is thus a valuable tool for high-resolutionimaging of biological or nonbiological samples. The STORM concept isalso applicable to other photoswitchable fluorophores and fluorescentproteins, which will potentially allow high-resolution live-cell imagingwith endogenous labels, and any other imaging applications in whichsub-diffraction limit image resolution is desired.

For additional details and examples, see W. M. Bates, et al.,“Multicolor Super-resolution Imaging with Photo-switchable FluorescentProbes” Science (in press), or M. J. Rust, et al.,“Sub-diffraction-limit imaging by stochastic reconstruction opticalmicroscopy (STORM),” Nature Methods 3, 793-795 (2006).

Following are methods useful in the above description.

Preparation of Switch-Labeled DNA Constructs.

Biotinylated and/or amine-modified DNA oligonucleotides (“oligos”) werepurchased, PAGE purified, from Operon. Amine-modified oligos werelabeled with amine reactive Cyanine dyes (Amersham Bioscience) or Alexadyes (Invitrogen) post-synthetically following the protocol provided bythe manufacturer. The dye-labeled oligos were purified using reversephase HPLC. Complementary strands of DNA were annealed to formbiotinylated double-stranded DNA (dsDNA) by mixing equimolar amounts ofthe two complementary strands in 10 mM Tris-Cl (pH 7.5), 50 mM NaCl,heating to 90° C. for two minutes, and then allowing the mixture to coolto room temperature in a heat block over a period of one hour.

Biotinylated dsDNA of varying lengths having an intra-switch distance of135 bp were constructed by annealing complimentary oligos as describedabove to form three different 45 bp dsDNA segments denoted A, B, and C,followed by a ligation reaction. The oligos were designed with specificsticky ends which only permit A to ligate to B, B to ligate to C and Cto ligate to A, reading in the 5′ to 3′ direction. One oligo (A)contained amine-reactive sites three base pairs apart on oppositestrands which were specifically labeled with Cy3 and Cy5 prior toannealing to form the optical switch. Oligos B and C contain twointernal biotin modifications per strand to facilitate multivalentlinkage to the streptavidin surface. After annealing, the oligos weremixed at equal concentrations and ligated overnight using T4 ligase (NewEngland Biolabs). The resulting ligation product was purified on a 1.5%agarose gel to select bands containing the desired concatamerized dsDNAlength.

Preparation of Switch-Labeled Antibodies.

Goat antimouse IgG secondary antibodies (Abcam or Invitrogen) and goatanti-rabbit antibody (Abcam) were labeled non-specifically withamine-reactive Cy2 or Cy3 (to serve as an activating portion) and Cy5 orAlexa 647 (to serve as a light-emitting portion). The averagedye-to-antibody ratio was ˜2:1 for Cy3 and ˜0.1:1 for Cy5 in the case ofRec A imaging. In the case of microtubule and clathrin imaging,dye-to-antibody ratio was ˜2:1 for Cy3 or Cy2 and ˜0.4:1 for Alexa Fluor647. Various Cy5-to-antibody or Alexa 647-to-antibody ratios may be usedfor STORM imaging, for example, ratios up to or greater than 0.8.Various Cy3-to-antibody ratios and Cy2-to-antibody ratios can also beused for efficiently STORM imaging. The imaging quality appears not tobe very sensitive the Cy3 (or Cy2)-to-antibody ratio. These labelingratios were chosen to minimize the fraction of antibodies labeled withmore than one Cy5 molecule, due to the inefficient switching observedfor antibodies labeled with multiple Cy5. The presence of more than oneCy3 molecule on a single antibody did not interfere with switching. Withthis labeling ratio, a significant fraction of the secondary antibodydid not carry Cy5, and were not observed. This lower density of labelsis typically not a problem for indirect immunofluorescence imaging and,in particular, did not prevent resolution of the circular structure ofthe RecA-plasmid filaments or microtubules.

Preparation of RecA Filaments.

Biotinylated RecA was prepared by reacting purified recombinant RecA(New England Biolabs) with amine-reactive biotin-XX (Invitrogen) in 0.1M carbonate buffer at pH 8.3. The resulting biotinylated protein waspurified on a NAP-5 size exclusion column (Amersham). RecA filamentswere formed on ΦXRF-II (Phi-XRF) plasmid DNA (New England Biolabs) byincubating RecA (25% biotinylated:75% unbiotinylated, concentration of80 micrograms/mL) with plasmid DNA (2 micrograms/mL) in 10 mM Trisbuffer at pH 7.0, 100 mM NaCl, 7 mM MgCl₂, and 0.8 mg/mL ATP-γ-S(ATP-gamma-S) for 1 hour at 37° C. The resulting RecA-DNA filaments werestored at 4° C. and used for imaging the same day.

Microscope Slide Preparation.

Quartz microscope slides (G. Finkenbeiner) were cleaned using Alconoxdetergent, followed by sonication for 15 minutes in acetone, 1 M aqueousKOH, ethanol, 1 M aqueous KOH, sequentially. Slides being prepared forlipid bilayers were submerged into a 5% HF solution for 2 hours afterthe second KOH step. Finally, the slides were rinsed with deionizedwater, and flame dried. The flow channels were prepared using two piecesof double-sided adhesive tape (3M) and covered with a No. 1.5 glasscoverslip (VWR).

Oxygen Scavenging System.

All imaging buffers were supplemented with the oxygen scavenging system,which included 10% (w/v) glucose (Sigma), 0.1% (v/v)beta-mercaptoethanol (Sigma), 500 micrograms/mL glucose oxidase (Sigma),and 10 micrograms/mL catalase (Roche). The oxygen scavenging system wasimportant for reliable photoswitching of the fluorophores. As low as0.01% of beta-mercaptoethanol can be used in some instances forefficiently photoswitching of the cyanine dyes. The beta-mercaptoethanolcan also be replaced by other reducing reagents such as glutathione andcysteins, in other embodiments.

Surface-Immobilization of DNA and Antibodies.

To immobilize the labeled dsDNA on a surface, quartz microscope slides(G. Finkenbeiner) were cleaned using Alconox detergent, sonicated in 1 MKOH, ethanol, and 1 M KOH sequentially before being rinsed with MilliQwater and flame dried. A biotinylated bovine serum albumin (BSA, Sigma)solution (1.0 mg/mL) was first added to the slides, followed by 0.25mg/mL streptavidin (Invitrogen), and finally the DNA sample at a lowconcentration (˜30 pM) in order to obtain a low surface density of DNAmolecules such that individual molecules were well separated andoptically resolvable. The slides were rinsed prior to the addition ofeach reagent.

To immobilize DNA constructs for the 3-color STORM imaging, threedifferent DNA constructs, each labeled with an Alexa 405-Cy5 pair, aCy2-Cy5 pair, or a Cy3-Cy5 pair were mixed in solution andco-immobilized onto a quartz slide as described above. A concentrationof 500 pM of DNA was used to reach a high surface density of immobilizedmolecules.

To immobilize the DNA sample labeled with multiple switches on a quartzslide, a lipid bilayer was first formed on the slide by flowing inliposomes of egg PC and 5% biotin-PE (Avanti). The liposomes were formedaccording to the manufacturer's instructions, extruded through a 0.05micrometer filter membrane at a concentration of 5 mg lipids/mL in DIwater, and then mixed with a 1:1 ratio with a buffer containing 10 mMTris at pH 8.0, 100 mM NaCl immediately before use. After a 2 hourincubation the bilayer was rinsed extensively with 50 mM Tris at pH 8.0,10 mM NaCl, and the bilayer was incubated with streptavidin (0.25 mg/mL,Invitrogen) for 30 minutes. After extensive washing, the streptavidinsurface was crosslinked in 4% v/v formaldehyde in PBS for 1 hour andrinsed with Tris buffer before allowing the biotinylated, switch-labeledDNA to bind. DNA was imaged in Tris buffer (50 mM Tris-Cl at pH 7.5, 10mM NaCl) with the oxygen scavenging system described above.

To immobilized switch-labeled antibodies on a surface, the quartz slideswere cleaned as described and incubated for 5 minutes with mouseanti-transferrin IgG (Abcam) allowing it to bind non-specifically. Theslide was then incubated with the labeled secondary antibodies in PBSbuffer containing 3% bovine serum albumin (BSA) for 10 min. The bufferwas replaced with 50 mM Tris, 10 mM NaCl, pH 7.5 containing the oxygenscavenging system for imaging.

Immunofluorescence Imaging of RecA-dsDNA Filaments.

Biotinylated RecA-dsDNA filaments were attached via streptavidinlinkages to a quartz slide non-specifically coated with biotinylatedBSA. The surface was then washed with 50 mM Tris at pH 7.0, 100 mM NaCl,7 mM MgCl₂, 3% BSA w/v (block buffer) and incubated for 30 minutes toblock the surface against non-specific antibody binding. The slide wasthen incubated in this block buffer containing monoclonal mouse antibodyagainst RecA (Stressgen) at 2 microgram/mL for 1 hour. After extensivewashing with block buffer, the slide was incubated in the block buffercontaining switch-labeled secondary antibody at 0.3 microgram/mL for 1hour. Finally, the sample was washed and imaged in 50 mM Tris at pH 7.5,100 mM NaCl, 7 mM MgCl₂, supplemented with the oxygen scavenging systemas described above.

Immunofluorescence imaging of microtubules and clathrin-coated pits incells. Green monkey kidney BS-C-1 cells were plated in LabTek II 8 wellchambered coverglass (Nunc) at a density of 30 k per well. After 16 to24 hr, they were rinsed with phosphate buffered saline (PBS) buffer,fixed with 3% formaldehyde, and 0.1% glutaraldehyde at room temperaturein PBS for 10 minutes, and quenched with 0.1% sodium borohydride in PBSfor 7 minutes to reduce the unreacted aldehyde groups and fluorescentproducts formed during fixation. The sodium borohydride solution wasprepared immediately before use to avoid hydrolysis. The fixed samplewas permeablized in blocking buffer (3% BSA, 0.5% Triton X-100 in PBS)for 10 min, stained with one or both of the primary antibodies againsttubulin and clathrin (2.5 micrograms/mL mouse anti-beta ((3) tubulin,ATN01 from Cytoskeleton and/or 2 micrograms/mL rabbit anti-clathrinheavy chain, ab21679 from Abcam) for 30 min in blocking buffer. Thesample was then rinsed with washing buffer (0.2% BSA, 0.1% Triton X-100in PBS) three times. Corresponding secondary antibodies labeled withphotoswitchable probes (2.5 micrograms/mL) were added to the sample inblocking buffer and then thoroughly rinsed after 30 minutes. Cellimaging was performed in a standard imaging buffer that contained 50 mMTris, pH 7.5, 10 mM NaCl, 0.5 mg/mL glucose oxidase (Sigma, G2133), 40micrograms/mL catalase (Roche Applied Science, 106810), 10% (w/v)glucose and 1% (v/v) beta-mercaptoethanol. Beta-mercaptoethanol wasfound to be important for the observed photoswitching behavior of Cy5,Cy5.5, and Cy7, but even a low concentration of beta-mercaptoethanol (aslow as 0.02% v/v, or potentially lower) supported photoswitching.Beta-mercaptoethanol at low concentrations (0.1%) was compatible withlive cell imaging. Photoswitching was also observed whenbeta-mercaptoethanol was replaced with cysteine (100 mM), which was alsocompatible with live cell imaging. Glucose oxidase was used as an oxygenscavenger system to increase the photostability of the cyanine dyes, andcells were viable at the reported glucose oxidase concentration for atleast 30 minutes.

Goat anti-mouse antibody (Invitrogen) and goat anti-rabbit antibody(Abcam) were each labeled with a mixture of amine-reactive activatorsand reporters. Alexa 405, Cy2, and Cy3 were used as the activatingportion of the photoswitch. Alexa 647 (Invitrogen), which has verysimilar structural and optical properties as Cy5, was used as thelight-emitting portion of the photoswitch. The concentrations of thereactive dyes were controlled such that each antibody had, on average,two activating dyes and 0.3-0.4 light-emitting dyes.

Imaging Procedures.

Photoswitch characterization, DNA concatamer imaging and RecA-dsDNAimaging were performed with an Olympus IX71 microscope. Single-moleculeimaging was conducted in the prism-type total internal-reflectionfluorescence (TIRF) imaging geometry. The samples were excited with a657 nm laser, and activated with a 532 nm, a 457 nm or a 405 nm laser.The fluorescence emission of the dyes was collected with a N.A. 1.25 60×water immersion objective, and imaged onto an electron multiplying CCDcamera (Andor Ixon DV897) after passing through a 665 nm long passfilter (Chroma). To track motion of the sample stage, 200 nm redfluorescent polystyrene beads (Invitrogen, F-8810) were added to theslide in Tris buffer containing 10 mM MgCl₂ and allowed to bind to thesurface. Data was acquired using custom data acquisition softwarewritten in Labview, which enabled sequences of alternating red and greenlaser excitation pulses to be applied to the sample, switching the dyeson and off. Laser excitation was synchronized with the camera exposureto 1 ms accuracy.

Imaging experiments on DNA concatamers typically included of 60 laserpulse cycles to activate and deactivate the switches, where each cyclelasted for 5 s, so that the final image took 5 minutes to acquire. Themultiple cluster configuration of the centroid position distribution istypically apparent within 2 minutes. The experiments on antibody-labeledRecA-dsDNA filaments included 70 switch cycles, each lasting 10 s. Thering shape of the RecA-dsDNA filaments typically became evident within 2minutes, although the position of every switch present in the sample hadnot yet been identified by this time. As the rate of switching dependedlinearly on the excitation intensity, the cycling time, and hence theoverall imaging time, could be shortened without substantially affectingthe localization accuracy by increasing the red laser intensity. Thegreen laser intensity was chosen so that typically 1-3 switches wereswitched on during the green pulse and, in most cases, all activatedswitched were turned off during the red phase of the cycle.

Raw images of RecA-dsDNA for comparison with STORM reconstructed imageswere formed by taking the maximum fluorescence value recorded for eachpixel throughout the imaging sequence so that each switch would beequally represented regardless of the amount of time it spent in thefluorescent or dark states.

Three-color STORM imaging of the DNA sample was performed on an OlympusIX71 inverted microscope in the prism-type TIRF configuration. A 633 nmHeNe laser was used as the imaging laser and the violet (405 nm), blue(457 nm), and green (532 nm) lasers were used as the activation lightsources. The sample was first exposed to the red imaging light to switchoff nearly all Cy5 dyes in the field of view. Then the sample wasperiodically activated with a sequence of violet, blue, and green laserpulses each of which switched on a sparse, optically resolvable subsetof fluorophores which were then imaged with the red laser. Fluorescencefrom these probes was detected with a CCD camera after passing through along pass emission filter (Chroma, HQ645LP). During the STORM dataacquisition, the camera recorded the fluorescence signal at a constantframe rate of 19 Hz. In each switching cycle, one of the activationlasers was turned on for 1 frame, followed by 9 frames of illuminationwith the red imaging laser.

STORM imaging of cells was performed on the Olympus IX71 microscope withan objective-type TIRF imaging configuration. A custom polychroicbeamsplitter (z458/514/647rpc, Chroma) reflected the excitation laserlight onto the sample through an objective (100× oil, NA 1.4, UPlanSApo,Olympus), and fluorescence emission from the sample was collected by thesame objective. Emitted light was filtered with two stacked dual-bandemission filters (51007m, Chroma, and 595-700DBEM, Omega Optical) beforebeing imaged on the EMCCD camera. The use of a dual-band emitter enablesfluorescence from Cy3 to be collected in addition to the fluorescence ofthe reporter dyes. Cy3 fluorescence collected during frames in which thegreen activation laser was on was used for drift correction purposes andto generate the conventional fluorescence image. For single-color STORMimaging with Alexa 647 as the light emitting portion of the photoswitchand Cy3 as the activating portion, the red laser (657 nm) was used forimaging and green (532 nm) laser pulses were for activation. Fortwo-color STORM imaging with Cy2 and Cy3 as the activating portion,alternating blue and green (457 and 532 nm) laser pulses were used foractivation. Images were acquired at a frame rate of 19 Hz. In eachswitching cycle, one of the activation lasers was turned on for 1 frame,followed by 9 frames of illumination with the red imaging laser. Typicallaser powers used for STORM imaging were 40 mW for the red laser and 2microwatts for each of the activation lasers.

Image Analysis.

In one example of image analysis, fluorescent structures in an averagedimage were first isolated in 13×13 pixel square fitting window for dataanalysis. In a given window, the total fluorescence intensity wasintegrated in each frame to produce a fluorescence time trace (see FIG.6). Several criteria were used to ensure high accuracy localization ofsingle switches:

(1) In each switching cycle, only regions where a single switch is onfor at least three frames (0.3 seconds) were used for localizationanalysis (see FIG. 6).

(2) The fluorescence images within these regions were fit by nonlinearleast-squares regression to a continuous ellipsoidal Gaussian:

I(x,y)=A+I ₀ e ^([−(x′/a)) ² ^(−(y′/b)) ² ^(]/2)

where:

x′=(x−x ₀)cos θ−(y−y ₀)sin θ

y′=(x−x ₀)sin θ+(y−y ₀)cos θ

Here, A is the background fluorescence level, I₀ is the amplitude of thepeak, a and b reflects the widths of the Gaussian distribution along thex and y directions, x₀ and y₀ describe the center coordinates of thepeak, and θ (theta) is the tilt angle of the ellipse relative to thepixel edges. Based on this fit, the peak ellipticity defined as|2(a−b)/(a+b)| was computed. If this ellipticity exceeded 15%,indicating poor image quality or the possible presence of multipleactive switches, the region was rejected from the analysis.

(3) The total number of counts collected in the peak was calculated as2π ab I₀ (2 pi ab I₀) and then converted to photoelectrons, and thus thenumber of photons detected, using the camera manufacturer's calibratedcurve for the electron multiplication and ADC gain settings used duringimaging. If the total number of photoelectrons in the peak was less than2,000, the region was rejected due to insufficient statistics to achievehigh localization accuracy.

The regions of the fluorescence traces that passed the above tests wereused for the final localization analysis. The fluorescence imagescorresponding to these regions were subjected to a final fit using apixelated Gaussian function to determine the centroid position. Becausethe CCD chip included square pixels of finite size, for optimumaccuracy, the image was fit to:

${I\left( {x,y} \right)} = {A + {\int_{x - \delta}^{x + \delta}{{dX}{\int_{y - \delta}^{y + \delta}{{dY}\mspace{14mu} I_{0}e^{{\lbrack{{- {(\frac{X - x_{0}}{a})}^{2}} - {(\frac{Y - y_{0}}{b})}^{2}}\rbrack}/2}}}}}}$

which, for ease of evaluation, can be re-expressed in terms of errorfunctions as:

${I\left( {x,y} \right)} = {A + {I_{0}{{\frac{a\; b\; \pi}{4}\begin{bmatrix}{{{erf}\left( \frac{x + \delta - x_{0}}{a} \right)} -} \\{{erf}\left( \frac{x - \delta - x_{0}}{a} \right)}\end{bmatrix}}\left\lbrack \begin{matrix}{{{erf}\left( \frac{y + \delta - y_{0}}{b} \right)} -} \\{{erf}\left( \frac{y - \delta - y_{0}}{b} \right)}\end{matrix} \right\rbrack}}}$

where A, I₀, a, b, x₀ and y₀ are as defined previously and δ(delta) isthe fixed half-width of a pixel in the object plane. The final centroidcoordinates (x₀, y₀) obtained from this fit were used as one data pointin the final STORM image.

In another example of image analysis, an image movie was prepared thatincluded a repetitive sequence of activation frames (in which theactivation laser is on) and imaging frames (in which the imaging laseris on). For each imaging frame, fluorescent spots were identified andfit to Gaussian and/or elliptical Gaussian functions to determine theircentroid positions, intensities, widths, and ellipticities. Based onthese parameters, peaks too dim, too wide or too skewed to yieldsatisfactory localization accuracy were rejected from further analysis.Peaks appearing in consecutive imaging frames with a displacementsmaller than one camera pixel were considered to originate from the samefluorescent molecule and centroid positions of these peaks wereconnected across frames and organized into a data structure, which isreferred to as a “string.” Each string represents a single switchingcycle for one fluorescent reporter molecule: the starting point of thestring is the frame in which the molecule is switched on and itsendpoint is the frame in which the molecule switches off. The finallocalization of the molecule was determined as the weighted average ofthe centroid positions across the entire string, weighted by the peakintensity of each frame. The total number of photons detected for eachswitching cycle was used as an additional filter to further rejectlocalizations with low accuracy. Strings starting in an imaging frameimmediately after an activation frame were recognized as a controlledactivation event and color-coded according to the activation lasercolor. Other strings were identified as non-specific activations, mostlikely induced by the red imaging laser.

To correct for mechanical drift in the microscope during imaging, thesame fitting algorithm was used to automatically track the motion ofseveral fluorescent beads in the field of view. The beads served asfiducial marks and their positions were sampled during each imagingcycle. The averaged motion of the beads was subtracted from thecoordinates obtained for each single switch position, yielding adrift-corrected reconstructed image.

As another drift correction method, the activator fluorophores wasimaged during the activation frame and the correlation function wascalculated between the first activation frame and all subsequentactivation frames. By tracking the centroid of the correlation function,the drift of the image could be determined and corrected for in theSTORM image. The correlation functions obtained from the fiducial markerimages may also be used for drift correction. In some cases, it was alsofound that some further drift correction was possible by analyzing thecorrelation function of the STORM image itself as a function of time.

For quantitative analysis of images using switches equally spaced ondsDNA, the coordinates in the reconstructed image were classified usinga k-means clustering algorithm, and inter-switch distances werecalculated as the distance between the cluster centroids. The number ofclusters input to the algorithm was chosen according to the number ofphotobleaching steps observed during the initial exposure to red light.

Prediction of DNA Configuration.

Possible configurations of a 135 bp piece of dsDNA bound to the surfacewere computed by Monte Carlo simulation of the DNA as a worm-like chainin the plane. According to the Watson-Crick structure of dsDNA andaccounting the length of the C₆ linkers attaching the Cy5 molecules tothe nucleotides, the expected contour length between neighboring Cy5molecules of 46±1 nm was calculated. The DNA was treated as a series of1 A (Angstrom) joints lying in the plane, each of which was deflected bya random angle selected from a Gaussian distribution chosen to give apersistence length of 50 nm. The measured inter-switch spacing wascompared to the distribution of end-to-end distances of the polymer inthis simulation. Single-molecule imaging of photoswitchableactivator-reporter pairs. To characterize the switching kinetics of thephotoswitchable probes from above, the two fluorescent dye molecules(activator and reporter) were conjugated to the end of a double strandedDNA (dsDNA) construct, and the construct was immobilized on a quartzsurface for single-molecule imaging. The DNA constructs were labeled asfollows. Briefly, PAGE purified DNA oligonucleotides with biotin and/oramine modification at the ends were obtained from Operon. The oligos (30base pairs (bp) in length) were labeled with amine reactive dyes (Cy2,Cy3, Cy5, Cy5.5, and Cy7 were obtained from GE Healthcare, and AlexaFluor 405 and Alexa Fluor 647 were obtained from Invitrogen)post-synthetically following the protocol provided by the manufacturers.The dye-labeled oligos were purified using reverse phase HPLC.Complementary strands of DNA, each labeled with an activator or areporter dye, were annealed to form biotinylated dsDNA by mixingequimolar amounts of the two complementary strands in 10 mM Tris-Cl (pH7.5), 50 mM NaCl. This allowed a pair of activator and reporter dyes tobe brought into close proximity, as illustrated in FIG. 13H,facilitating the immobilization of dye pair to a microscope slide viabiotin-straptavidin linkage FIG. 13H is a schematic of double-strandedDNA and antibody molecules labeled with a Cy3-Cy5 pair.

To immobilize the labeled dsDNA on a surface, quartz microscope slides(G. Finkenbeiner) were cleaned using Alconox detergent, sonicated in 1MKOH, ethanol, and 1M KOH sequentially before being rinsed with MilliQwater and flame dried. A biotinylated bovine serum albumin (b-BSA,Sigma) solution (1.0 mg/mL) was first added to the slides, followed by0.25 mg/mL streptavidin (Invitrogen), and finally the DNA sample at alow concentration (˜30 pM) in order to obtain a low surface density ofDNA molecules such that individual molecules were well separated andoptically resolvable from each other. The slides were rinsed prior tothe addition of each reagent. Single-molecule imaging was performed in astandard imaging buffer that contains 50 mM Tris, pH 7.5, 10 mM NaCl,0.5 mg/mL glucose oxidase (Sigma, G2133), 40 micrograms/mL catalase(Roche Applied Science, 106810), 10% (w/v) glucose and 1% (v/v)beta-mercaptoethanol.

Single-molecule imaging was performed on an Olympus IX-71 invertedmicroscope equipped with prism-type total internal reflectionfluorescence (TIRF) configuration. A red 657 nm diode laser(RCL-200-656, Crystalaser) was used to excite fluorescence from thereporter fluorophore and to switch them off to the dark state. A 532 nmdiode-pumped solid state laser (GCL-200-L, Crystalaser), the 457 nm lineof an Ar ion laser (35-LAL-030-208, Melles Griot), and a 405 nm diodelaser (CUBE 405, Coherent) were used to reactivate the reporters byexciting the different activators. The fluorescence signal from thereporter dyes was collected by a 60×, NA 1.2 water immersion objective(Olympus) and then imaged on to an EMCCD camera (Andor Ixon DV897DCS-BV)after passing through a band pass fluorescence emission filter (Chroma,HQ710/80m for Cy5 and Cy5.5 and HQ740LP for Cy7). A 1.6× tube lens wasused to set the final imaging magnification to ˜100×.

Switching Kinetics Analysis of the Photoswitchable Dyes.

To measure switching kinetics, the DNA samples were first illuminatedwith the red imaging laser (657 nm) to switch the reporter moleculesinto the dark state, and the rate at which they switched off (k_(off))was measured by recording the number of fluorescent molecules as afunction of time and fitting it to a single exponential function. Formeasurements of k_(on), after switching the reporter fluorophores offand while the red imaging laser remained on, the sample was exposed tothe activation laser (405 nm, 457 nm, or 532 nm), which caused thefluorophores to switch back on, reaching equilibrium between activationand deactivation. The number of fluorophores in the fluorescent state atequilibrium was measured, and the activation rate constant (k_(on)) wasthen calculated from the independently determined value of k_(off) andthe fraction of molecules (F) in the fluorescent state at equilibrium,according to the relation F=k_(on)/(k_(on)+k_(off)).

Photon Number Analysis of the Photoswitchable Dyes.

The number of photons detected per switching cycle for Cy5, Cy5.5, andCy7 were measured when they were paired with Cy3 as the activator on DNAand antibody molecules. The average number of photons detected perswitching cycle was a constant independent of the excitation laserintensity. The photon number, however, depended on the emission filtersand imaging geometry used. Using the prism-type TIRF imaging geometry,657 nm imaging laser, and two stacked HQ665LP emission filters for Cy5and Cy5.5 and a HQ740LP emission filter for Cy7, the photon numbersdetected were ˜3000 for Cy5 and Cy5.5 and −500 for Cy7. These numberscorrespond to a theoretical limit of localization accuracy (in terms ofstandard deviation or s.d.) of 3 nm for Cy5 and Cy5.5 and 9 nm for Cy7,calculated using the formula

${s.d.} = {\sqrt{{\left( {S^{2} + {a^{2}/12}} \right)/N} + {4\sqrt{\pi}S^{3}{b^{2}/a}\; N^{2}}}.}$

In the formula, S is the standard deviation of the point spread functionof the imaging setup, a is the edge size of the area imaged on each CCDpixel, b is the background noise level, and N is the number of photonsdetected (S=173 nm for Cy5/Cy5.5 and =200 nm for Cy7, a=165 nm, b=6 forCy5/Cy5.5 and =1 for Cy7).

In this work, full-width-half-maximum (FWHM) was typically used todescribe imaging resolution. The FWHM values corresponding to thelocalization accuracies quoted above are 8 nm for Cy5 and Cy5.5 and 22nm for Cy7. Using the objective-type TIRF imaging geometry, 657 nmimaging laser, and stacked HQ665LP and HQ710/70BP emission filters forCy5 and Cy5.5 and stacked HQ740LP and 800WB80 emission filters for Cy7,the photon numbers detected were ˜6000 for Cy5 and Cy5.5 and ˜1000 forCy7, which were approximately twice as high as the numbers obtained inthe prism-type TIRF geometry. The number of photons detected for Alexa647, a cyanine dye with a similar structure to that of Cy5 (see FIG.13A), was within 10% of the number detected from Cy5. FIGS. 13A-13Dillustrate structures of the photoswitchable reporters Cy5, Alexa 647,Cy5.5, and Cy7. “R” stands for the place where DNA or antibody wasattached. The photon numbers detected from theactivator-reporter-labeled DNA samples were slightly smaller than thenumbers detected from the corresponding antibody samples. The HQ665LP,HQ740LP, and HQ710/70BP filters were obtained from Chroma and the800WB80 filter was from Omega.

FIGS. 13E-13F illustrate normalized absorption and emission spectra ofCy5, Alexa 647, Cy5.5, and Cy7 in aqueous solution. The absorptionspectra were normalized by the maximum absorption value, and theemission spectra were normalized by the integrated peak area. FIG. 13Gillustrates normalized absorption spectra of activator dyes, Alexa 405,Cy2, and Cy3 in aqueous solution. FIG. 14 illustrates photoswitchingbehavior of the Alexa 405-Cy7 pair. The lower panel shows a fluorescencetime trace of Cy7. The upper panel shows the 405 nm laser pulses used toactivate the dye pair. A red laser (657 nm) was continuously on, servingto excite fluorescence from the Cy7 and to switch it off to the darkstate.

FIG. 15 shows a conventional fluorescence image of a mixture of threedifferent DNA constructs, each labeled with Cy3-Cy5, Cy2-Cy5, or Alexa405-Cy5 and mixed at a high surface density on a microscope slide. Thefluorescence image was taken from all of the Cy5 molecules in thisregion before the sample was subjected to any photoswitching. A thermalcolor scheme is used here to illustrate the intensity, with blackindicating low intensity, red higher, and yellow highest. A three-colorimage of the same region is shown in FIG. 12A. The overall intensityprofile may appear to be slightly different for the two images due tothe different numbers of switching cycles exhibited by individualmolecules.

Three-Color Imaging of a Model DNA Sample.

Three different DNA constructs, each labeled with an Alexa 405-Cy5 pair,a Cy2-Cy5 pair, or a Cy3-Cy5 pair were mixed in solution andco-immobilized onto a quartz slide as described above. A concentrationof 500 pM of each DNA was used to reach a high surface density ofimmobilized molecules. Due to a moderate Cy5 quenching effect thatoccurred when a Cy3 molecule was positioned in very close proximity, inthis experiment these two dyes were separated by 9 base pairs on a 43 bpdsDNA, instead of being attached to the end of a dsDNA. This Cy5quenching effect was less significant when Alexa 405 or Cy2 waspositioned in very close proximity. Fluorescent beads (Molecular probes,F8801) were added to the sample slide as fiducial markers for thepurpose of drift correction.

Imaging was performed on an Olympus IX71 inverted microscope in theprism-type TIRF configuration. A 633 nm HeNe laser (25-LHP-928-249,Melles Griot) was used as the imaging laser and the violet (405 nm),blue (457 nm), and green (532 nm) lasers mentioned above were used asthe activation light sources. The sample was first exposed to the redimaging light to switch off nearly all Cy5 dyes in the field of view.Then the sample was periodically activated with a sequence of violet,blue, and green laser pulses each of which switched on a sparse,optically resolvable subset of fluorophores which were then imaged withthe red laser. Fluorescence from these probes was detected with the CCDcamera after passing through a long pass emission filter (Chroma,HQ645LP). During data acquisition, the camera recorded the fluorescencesignal at a constant frame rate of 19 Hz. In each switching cycle, oneof the activation lasers was turned on for 1 frame, followed by 9 framesof illumination with the red imaging laser. Under typical imagingconditions, an average fluorophore remains in the fluorescent state forthree frames after activation, and ˜3000 photons per molecule weredetected during each switching cycle.

Imaging of Microtubules and Clathrin-Coated Pits in Cells.

Green monkey kidney BS-C-1 cells were plated in LabTek II 8 wellchambered coverglass (Nunc) at a density of 30K per well. After 16 to 24hr, cells were rinsed with phosphate buffered saline (PBS) buffer, fixedwith 3% formaldehyde, and 0.1% glutaraldehyde at room temperature in PBSfor 10 min, and quenched with 0.1% sodium borohydride in PBS for 7 minto reduce the unreacted aldehyde groups and fluorescent products formedduring fixation. The sodium borohydride solution was preparedimmediately before use to avoid hydrolysis. The fixed sample waspermeablized in blocking buffer (3% BSA, 0.5% Triton X-100 in PBS) for10 min, stained with one or both of the primary antibodies againsttubulin and clathrin (2.5 micrograms/mL mouse anti-beta tubulin, ATN01from Cytoskeleton and 2 micrograms/mL rabbit anti-clathrin heavy chain,ab21679 from Abcam) for 30 min in blocking buffer. The sample was thenrinsed with washing buffer (0.2% BSA, 0.1% Triton X-100 in PBS) threetimes. Corresponding secondary antibodies labeled with photoswitchableprobes (2.5 micrograms/mL) were added to the sample in blocking bufferand then thoroughly rinsed after 30 min. Cell imaging was performed in astandard imaging buffer that contains 50 mM Tris, pH 7.5, 10 mM NaCl,0.5 mg/mL glucose oxidase, 40 micrograms/mL catalase, 10% (w/v) glucoseand 1% (v/v) beta-mercaptoethanol. It was found thatbeta-mercaptoethanol was important for the observed photoswitchingbehavior of Cy5, Cy5.5, and Cy7, but even low concentrations ofbeta-mercaptoethanol (as low as 0.02% v/v) supported photoswitching.Beta-mercaptoethanol at low concentrations (0.1% and 0.02%) wascompatible with live cell imaging. Photoswitching was also observed whenbeta-mercaptoethanol was replaced with cysteine (100 mM), which was alsocompatible with live cell imaging. Glucose oxidase was used as an oxygenscavenger system to increase the photostability of the cyanine dyes, andcell morphology was normal at the reported glucose oxidase concentrationfor at least 30 min. In this work, all imaging experiments wereperformed on fixed cells.

Goat anti-mouse antibody (Invitrogen) and goat anti-rabbit antibody(Abcam) were each labeled with a mixture of amine-reactive activatorsand reporters. Cy2 and Cy3 were used as the activators. Alexa 647(Invitrogen), which has similar structural and optical properties to Cy5(FIG. 13), was used as the reporter. The concentrations of the reactivedyes were controlled such that each antibody had, on average, twoactivator molecules and 0.3 to 0.4 reporter molecules. Thephotoswitching behavior was relatively insensitive to the number ofactivators per antibody. The labeling ratio of two activators perantibody was chosen to ensure that the majority of antibodies hadactivators and thus to optimize the staining efficiency. However, whenmore than one reporter molecule was attached to the same antibody, itwas found that the close proximity of the reporter molecules lowered theoff rate. To assess this effect more quantitatively, dsDNA moleculeslabeled with two Cy5 dyes of known separations were prepared. The offrate of the construct having two Cy5 dyes separated by 2 nm was ˜5 timesslower than that for a construct with a single Cy5. For constructs wherethe two Cy5 dyes were separated by 7 nm or 14 nm, the off rates wereroughly comparable to that of the single-Cy5 construct. Thisself-interaction effect was slightly less pronounced for Alexa 647 ascompared with Cy5. Practically, when labeling antibody, a relatively lowdye/protein ratio was chosen here for the reporter (0.3 to 0.4) suchthat the majority of reporter-labeled antibody molecules have only onereporter.

Imaging was performed on the Olympus IX71 microscope with anobjective-type TIRF configuration. A custom polychroic beamsplitter(z458/514/647rpc, Chroma) reflected the excitation laser light onto thesample through an objective (100× oil, NA 1.4, UPlanSApo, Olympus), andfluorescence emission from the sample was collected by the sameobjective. Emitted light was filtered with two stacked dual-bandemission filters (51007m, Chroma, and 595-700DBEM, Omega optical) beforebeing imaged on the EMCCD camera. The use of a dual-band emitter enablesfluorescence from Cy3 to be collected in addition to the fluorescence ofthe reporter dyes. Cy3 fluorescence collected during frames in which thegreen activation laser was on was used for drift correction purposes andto generate the conventional fluorescence image. For single-colorimaging with Alexa 647 as the reporter and Cy3 as the activator, the redlaser (657 nm) was used for imaging and green (532 nm) laser pulses werefor activation. For two-color imaging with Cy2 and Cy3 as theactivators, alternating blue and green (457 and 532 nm) laser pulseswere used for activation. Images were acquired at a frame rate of 19 Hz.In each switching cycle, one of the activation lasers was turned on for1 frame, followed by 9 frames of illumination with the red imaginglaser. Because the two stacked dual-band emission filters (51007m and595-700DBEM) significantly cut fluorescence signal from Alexa 647, only˜3000 photons, instead of ˜6000, were detected on average from oneantibody during each switching cycle. Typical laser powers used forimaging were 40 mW for the red laser and 2 microwatts for each of theactivation lasers.

Image Analysis.

A typical image was generated from a sequence of 2000 to 100000 imageframes recorded at 19 Hz. The movie included a repetitive sequence ofactivation frames (in which the activation laser is on) and imagingframes (in which the imaging laser is on). For each imaging frame,fluorescent spots were identified and fit to a Gaussian or ellipticalGaussian function to determine their centroid positions, intensities,widths and ellipticities. Based on these parameters, peaks too dim, toowide or too elliptical to yield satisfactory localization accuracy wererejected from further analysis. Peaks appearing in consecutive imagingframes with a displacement smaller than one camera pixel were consideredto originate from the same fluorescent molecule, and centroid positionsof these peaks were connected across frames and organized into a datastructure which is referred to here as a “string.” Each stringrepresents a single switching cycle for one fluorescent reportermolecule: the starting point of the string is the frame in which themolecule is switched on and its endpoint is the frame in which themolecule switches off. The final localization of the molecule wasdetermined as the weighted average of the centroid positions across theentire string, weighted by the number of photons detected in each frame.The total number of photons detected for each switching cycle was usedas an additional filter to further reject localizations with lowaccuracy. Strings starting in an imaging frame immediately after anactivation frame were recognized as a controlled activation event andcolor-coded according to the activation laser color. Other strings wereidentified as non-specific activations, most likely induced by the redimaging laser as the amount of non-specific activation was observed toincrease with the red laser intensity (data not shown). Nonspecificactivation by the red imaging laser would also occur in the firstimaging frame and be counted as a controlled activation event, givingone source of error for color crosstalk.

Besides the number of photons detected in one imaging cycle, anotherfactor that limits the localization accuracy was sample drift during thecourse of the experiment. The drift was corrected by two methods. Thefirst method involved adding fiducial markers (fluorescent beads) totrack the drift of the sample and subtracting the movement of themarkers during image analysis. In the second method, the activatorfluorophores were imaged during the activation frame and calculated thecorrelation function between the first activation frame and allsubsequent activation frames. By tracking the centroid position of thecorrelation function, the drift of the image can be determined andcorrected for in the image. The correlation functions obtained from thefiducial marker images may also be used for drift correction. In somecases, it was found that further drift correction was possible byanalyzing the correlation function of the image itself as a function oftime.

For image presentation, each localization was assigned as one point inthe image. These points were either represented by a small marker (e.g.a cross) or rendered as a normalized 2D Gaussian peak, the width ofwhich was determined by its theoretical localization accuracy calculatedfrom the number of photons detected for that localization event. Formulticolor images, each localization was also false-colored according tothe color of the activation laser pulse. The following color codingscheme was typically used (although other coding schemes are possible):activations by the violet (405 nm) laser were shown in blue, those bythe blue (457 nm) laser were shown in green, and those by the green (532nm) laser were shown in red.

Crosstalk between different color channels resulted mainly from twoeffects: nonspecific activation and false activation. As describedearlier, nonspecific activations, mostly likely induced by the redimaging laser, can be most easily identified if the string did not startimmediately after an activation frame. However, such a nonspecificactivation may also occur during the frame immediately after anactivation laser pulse and thus be incorrectly assigned a color,although this mis-assignment will occur with a relatively lowprobability. Three methods can be used to reduce nonspecificactivation-induced crosstalk: (1) increasing the activation laserintensity, providing that the density of activated probes remains lowenough for single-molecule localization; (2) using a faster frame ratewhich effectively improves identification of those molecules activatedby the activation laser pulse; and (3) decreasing the imaging laserintensity to reduce the non-specific activation rate, but at the cost ofreducing imaging speed and/or accuracy. The second source of colorcrosstalk, false activations, stems from probes which were switched onby the wrong activation laser. Combining these two sources, the overallcrosstalk ratios under the typical cell imaging conditions used herewere measured to be 15% to 25% for the leakage of Cy2 signal into theCy3 channel and 25 to 35% for the leakage of Cy3 signal into the Cy2channel. For the three-color imaging of the DNA sample, crosstalkeffects were observed to be somewhat smaller because nonspecificactivation was observed to be less pronounced (FIG. 16), in part due tostronger activation laser powers used.

FIG. 16 shows crosstalk analysis for the three-color image of the DNAsample. The image shows separated clusters of localizations, eachcluster corresponding to an individual DNA molecule (FIGS. 12A-12C).Each of the localizations was colored according the activation laserused: localizations activated by the 405 nm laser were assigned the bluecolor, those activated by the 457 nm laser were assigned the green colorand those activated by the 532 nm laser were assigned the red color. Themajority of the localizations within each cluster displayed the samecolor, identifying the type of activator dye (Alexa 405, Cy2, or Cy3)present on the DNA molecule. The numbers of localizations of each colorwere counted for individual clusters and the fractions of localizationsassigned to each color channel are plotted here for the Alexa 405, Cy2;and Cy3 clusters. The crosstalk ratios can be calculated from the ratiosof incorrectly to correctly colored localizations.

The crosstalk ratios between different color channels under each imagingcondition can be quantitatively determined using samples singly labeledwith only one of the photoswitchable probes. Due to the clear separationbetween clathrin-coated pits and microtubules, the two color cell imageitself can also be used to estimate crosstalk quantitatively. Using thecrosstalk ratios, crosstalk can be effectively subtracted from amulticolor image. For instance, in the case of a two-color image, at anygiven location:

$\quad \left\{ \begin{matrix}{D_{1} = {d_{1} + {C_{2\rightarrow 1}d_{1}}}} \\{D_{2} = {{C_{1\rightarrow 2}d_{1}} + d_{2}}}\end{matrix} \right.$

where D₁ and D₂ are the observed local densities of spots in colorchannels 1 and 2, respectively, and d₁ and d₂ are the corresponding truelocal densities. C_(1→2) and C_(2→1) are the crosstalk ratios betweenthe two channels. The values of d₁ and d₂ can be solved from observedlocal densities D₁ and D₂ and crosstalk ratios C_(1→2) and C_(2→1). Thusthe probability of a localization at a given position in channel 1 beingassigned the wrong color is simply P₁=1−d₁/D₁. This point can thus beremoved according to this probability. Similar treatment can be appliedto every points in channels 1 and 2. To correct color crosstalk in thetwo-color images, a radius of 35 nm was chosen to calculate the localdensities. Due to the finite area required to reliably calculate localdensities, a slight erosion effect will arise from the crosstalksubtraction where two different colored structures overlap in space.According to simulations, for this example, if the imaging resolution is20 to 30 nm, such an operation will reduce the spatial resolution by˜20% when the crosstalk ratio is 20% for both channels. A similarstatistical approach can also be used to assign colors to nonspecificactivations (e.g. the probability of a non-specific activation belong tocolor channel 1 is d₁/(d₁+d₂), where d₁ and d₂ were obtained fromcontrolled activations as described above), effectively increasing theoverall localization point densities in the images, which may helpimprove resolution in cases where the resolution is point-densitylimited. Crosstalk subtraction and nonspecific activation colorassignment were applied in FIG. 11.

FIG. 17 shows localization accuracy for a single-color image of thecell. The localization accuracy was determined from point-like objectsin the cell, appeared as small clusters of localizations away from anydiscernable microtubule filaments. Shown here is the spatialdistribution of localizations within these point-like clusters. The 2Dhistogram of localizations was generated by aligning 170 clusters bytheir center of mass, each cluster containing more than 8 localizations.Fitting the 2D histogram with a Gaussian function gives a FWHM of 24 nm.

FIG. 18 shows localization accuracy for a two-color image of the cell.The localization accuracy was also determined from point-like objects inthe cell, appeared as small clusters of localizations away from anydiscernable microtubule or CCP structures. Shown here is the spatialdistribution of localizations within these point-like clusters. The 2Dhistograms of localizations were generated by aligning 187 clusters bytheir center of mass, each cluster containing more than 8 localizations.Fitting the 2D histogram with a Gaussian function gives a FWHM of 30 nm.

FIG. 19 shows images of clathrin-coated pits (CCPs). FIG. 19A shows acomparison of conventional fluorescence images (upper panels) and theimages generated here (lower panels). Nearly all CCPs appear to adopt aspherical structure. The rightmost panel shows two close-by CCPs thatwere resolved here, but appeared as a single nearly diffraction-limitedspot in the conventional fluorescence image. FIG. 19B shows the sizedistribution of 300 CCPs determined from the images as shown in FIG.19A.

While several embodiments of the present invention have been describedand illustrated herein, those of ordinary skill in the art will readilyenvision a variety of other means and/or structures for performing thefunctions and/or obtaining the results and/or one or more of theadvantages described herein, and each of such variations and/ormodifications is deemed to be within the scope of the present invention.More generally, those skilled in the art will readily appreciate thatall parameters, dimensions, materials, and configurations describedherein are meant to be exemplary and that the actual parameters,dimensions, materials, and/or configurations will depend upon thespecific application or applications for which the teachings of thepresent invention is/are used. Those skilled in the art will recognize,or be able to ascertain using no more than routine experimentation, manyequivalents to the specific embodiments of the invention describedherein. It is, therefore, to be understood that the foregoingembodiments are presented by way of example only and that, within thescope of the appended claims and equivalents thereto, the invention maybe practiced otherwise than as specifically described and claimed. Thepresent invention is directed to each individual feature, system,article, material, kit, and/or method described herein. In addition, anycombination of two or more such features, systems, articles, materials,kits, and/or methods, if such features, systems, articles, materials,kits, and/or methods are not mutually inconsistent, is included withinthe scope of the present invention.

All definitions, as defined and used herein, should be understood tocontrol over dictionary definitions, definitions in documentsincorporated by reference, and/or ordinary meanings of the definedterms.

The indefinite articles “a” and “an,” as used herein in thespecification and in the claims, unless clearly indicated to thecontrary, should be understood to mean “at least one.”

The phrase “and/or,” as used herein in the specification and in theclaims, should be understood to mean “either or both” of the elements soconjoined, i.e., elements that are conjunctively present in some casesand disjunctively present in other cases. Multiple elements listed with“and/or” should be construed in the same fashion, i.e., “one or more” ofthe elements so conjoined. Other elements may optionally be presentother than the elements specifically identified by the “and/or” clause,whether related or unrelated to those elements specifically identified.Thus, as a non-limiting example, a reference to “A and/or B”, when usedin conjunction with open-ended language such as “comprising” can refer,in one embodiment, to A only (optionally including elements other thanB); in another embodiment, to B only (optionally including elementsother than A); in yet another embodiment, to both A and B (optionallyincluding other elements); etc.

As used herein in the specification and in the claims, “or” should beunderstood to have the same meaning as “and/or” as defined above. Forexample, when separating items in a list, “or” or “and/or” shall beinterpreted as being inclusive, i.e., the inclusion of at least one, butalso including more than one, of a number or list of elements, and,optionally, additional unlisted items. Only terms clearly indicated tothe contrary, such as “only one of” or “exactly one of,” or, when usedin the claims, “consisting of,” will refer to the inclusion of exactlyone element of a number or list of elements. In general, the term “or”as used herein shall only be interpreted as indicating exclusivealternatives (i.e. “one or the other but not both”) when preceded byterms of exclusivity, such as “either,” “one of,” “only one of,” or“exactly one of.” “Consisting essentially of,” when used in the claims,shall have its ordinary meaning as used in the field of patent law.

As used herein in the specification and in the claims, the phrase “atleast one,” in reference to a list of one or more elements, should beunderstood to mean at least one element selected from any one or more ofthe elements in the list of elements, but not necessarily including atleast one of each and every element specifically listed within the listof elements and not excluding any combinations of elements in the listof elements. This definition also allows that elements may optionally bepresent other than the elements specifically identified within the listof elements to which the phrase “at least one” refers, whether relatedor unrelated to those elements specifically identified. Thus, as anon-limiting example, “at least one of A and B” (or, equivalently, “atleast one of A or B,” or, equivalently “at least one of A and/or B”) canrefer, in one embodiment, to at least one, optionally including morethan one, A, with no B present (and optionally including elements otherthan B); in another embodiment, to at least one, optionally includingmore than one, B, with no A present (and optionally including elementsother than A); in yet another embodiment, to at least one, optionallyincluding more than one, A, and at least one, optionally including morethan one, B (and optionally including other elements); etc.

It should also be understood that, unless clearly indicated to thecontrary, in any methods claimed herein that include more than one stepor act, the order of the steps or acts of the method is not necessarilylimited to the order in which the steps or acts of the method arerecited.

In the claims, as well as in the specification above, all transitionalphrases such as “comprising,” “including,” “carrying,” “having,”“containing,” “involving,” “holding,” “composed of,” and the like are tobe understood to be open-ended, i.e., to mean including but not limitedto. Only the transitional phrases “consisting of” and “consistingessentially of” shall be closed or semi-closed transitional phrases,respectively, as set forth in the United States Patent Office Manual ofPatent Examining Procedures, Section 2111.03.

What is claimed is: 1-211. (canceled)
 212. A system, comprising: anoptical system which provides activation light for activating a part ofa plurality of photoswitchable entities into a state able to emit lightand provides excitation light for exciting at least a part of theactivated entities; a detector which detects at least a part of lightemitted from the excited entities; and a controller, wherein thecontroller is programmed to calculate positional information of at leasta part of the plurality of entities by using information contained inthe detected light, wherein at least some of the entities are cyaninedyes.
 213. The system of claim 212, wherein the optical system comprisesa first optical system which provides activation light and a secondoptical system provides excitation light.
 214. The system of claim 213,wherein the activation light and the excitation light have substantiallythe same wavelength.
 215. The system of claim 213, wherein theactivation light and the excitation light have different wavelengths.216. The system of claim 212, wherein the cyanine dyes comprise at leastone of Cy5, Cy5.5, Cy7, Alexa Fluor® 647, Alexa Fluor® 405, Alexa Fluor®488, Cy2, Cy3, Cy3.5, Cy5, and a conjugate thereof.
 217. The system ofclaim 212, wherein the controller is programmed to correct thepositional information by using a fiduciary marker and movements of thefiduciary marker.
 218. The system of claim 217, wherein the correctionof the positional information comprises drift correction.
 219. Thesystem of claim 217, wherein the fiduciary marker is a fluorescentparticle.
 220. The system of claim 212, wherein the controller is ableto use Gaussian fitting to calculate the positional information. 221.The system of claim 212, wherein the controller is able to useelliptical Gaussian fitting to calculate the positional information.222. The system of claim 212, wherein the controller is able to generatean image by using the calculated positional information.
 223. The systemof claim 222, wherein the image is a high resolution image.
 224. Thesystem of claim 212, wherein the plurality of entities comprises a firstentity and a second entity.
 225. The system of claim 224, wherein: thefirst entity is activatable by a first wavelength of activation lightand the second entity is activatable by a second wavelength ofactivation light.
 226. The system of claim 224, wherein: the firstentity is excitable by a first wavelength of excitation light and thesecond entity is excitable by a second wavelength of excitation light.227. The system of claim 212, wherein the entities have a first portionand a second portion, the first portion being a light emitting portionand the second portion being an activator portion.
 228. The system ofclaim 212, wherein the controller is able to calculate positionalinformation of at least some of the plurality of entities to aresolution smaller than 20 nm.
 229. The system of claim 212, wherein thecontroller is able to use the positional information to construct animage, the image having a resolution that is better than thediffraction-limited resolution of the emitted light.
 230. The system ofclaim 212, wherein the controller is able to calculate the positionalinformation of the at least a part of the plurality of entities at morethan one point of time and/or as a function of time.
 231. The system ofclaim 212, wherein at least some of the plurality of entities areactivatable by light of different wavelengths and/or emit light atdifferent wavelengths.
 232. A program for causing a machine to perform aprocedure of calculating positional information performed by a system asclaimed in claim
 212. 233. A storage medium comprising a program forcausing a machine to perform a procedure of calculating positionalinformation performed by a system as claimed in claim
 212. 234. Anarticle, comprising a storage medium comprising a program for causing amachine to perform a procedure of calculating positional informationperformed by a system as claimed in claim
 212. 235. A method,comprising: applying activation light for activating a part of aplurality of photoswitchable entities into a state able to emit light,wherein the entities are cyanine dyes; applying excitation light forexciting at least a part of the activated entities; detecting at least apart of light emitted from the excited entities; and calculatingpositional information of at least a part of the plurality of entitiesby using information contained in the detected light.